N-type calcium channel antagonists for the treatment of pain

ABSTRACT

Compounds useful for the treatment of pain in accord with the following structural diagram,  
                 
 
wherein R 1 , R 2  and R 3  are any of a number of groups as defined in the specification and pharmaceutical compositions and methods of treatment utilising such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/415,853 filed Nov.3, 2003, which is the U.S. National Stage filing of InternationalApplication Serial No. PCT/SE01/02389 filed Oct. 31, 2001, which claimspriority to SE 0004054-3 filed Nov. 6, 2000, each of which isincorporated herein by reference in its entirety.

FIELD OF THE IVNENTION

This invention relates to compounds and methods for the treatment orprevention of pain or nociception.

RELATED ART

Pain causes a great deal of suffering and is a sensory experiencedistinct from sensations of touch, pressure, heat and cold. It is oftendescribed by sufferers by such terms as bright, dull, aching, pricking,cutting or burning and is generally considered to include both theoriginal sensation and the reaction to that sensation. This range ofsensations, as well as the variation in perception of pain by differentindividuals, renders a precise definition of pain difficult. Where painis “caused” by the stimulation of nociceptive receptors and transmittedover intact neural pathways, this is termed nociceptive pain. Pain mayalso be caused by damage to neural structures, and pain is often ismanifested as neural supersensitivity; this type of pain is referred toas neuropathic pain.

The level of stimulation at which pain is perceived is referred to asthe “pain threshold”. Where the pain threshold is raised, for instance,by the administration of an analgesic drug, a greater intensity or moreprolonged stimulus is required before pain is experienced. Analgesicsare a class of pharmaceutical agent which, following administration to apatient in need of such treatment, relieve pain without loss ofconsciousness. This is in contrast to other pain-relieving drugs, forexample, general anaesthetics which obtund pain by producing a hiatus inconsciousness, or local anaesthetics which block transmission inperipheral nerve fibres thereby preventing pain.

Tachykinin antagonists have been reported to induce antinociception inanimals, which is believed to be analogous to analgesia in man (forreview see Maggi et al, J. Auton. Pharmacol. (1993) 13, 23-93). Inparticular, non-peptide NK-1 receptor antagonists have been shown toproduce such analgesia, thus, for example, in classical tests ofchemo-nociception (phenylbenzoquinone-induced writhing and formalintest) the NK-1 receptor antagonist RP 67,580 produced analgesia withpotency comparable to that of morphine (Garret et al, Proc. Natl. Acad.Sci. USA (1993) 88, 10208-10212).

Opioid analgesics are a well-established class of analgesic agents.These compounds are generally accepted to include, in a generic sense,all drugs, natural or synthetic, with morphine-like actions. Thesynthetic and semi-synthetic opioid analgesics are derivatives of fivechemical classes of compound: phenanthrenes; phenylheptylamines;phenylpiperidines; morphinans; and benzomorphans. Pharmacologicallythese compounds have diverse activities, thus some are strong agonistsat the opioid receptors (e.g. morphine); others are moderate to mildagonists (e.g. codeine); still others exhibit mixed agonist-antagonistactivity (e.g. nalbuphine); and yet others are partial agonists (e.g.nalorphine). Whilst an opioid partial agonist such as nalorphine, (theN-alkyl analogue of morphine) will antagonise the analgesic effects ofmorphine, when given alone it can be a potent analgesic in its ownright. Of all of the opioid analgesics, morphine remains the most widelyused and is a suitable archetype compound. Unfortunately, apart from itsuseful therapeutic properties, morphine also has a number of drawbacksincluding respiratory depression, decreased gastrointestinal motility(resulting in constipation) and, in some individuals, nausea andvomiting may occur. Another characteristic is the development oftolerance and physical dependence which may limit the clinical use ofsuch compounds.

Anti-inflammatory compounds directed at blocking or reducing synovialinflammation, and thereby improving function, and analgesics directed toreducing pain, are presently the primary method of treating therheumatoid diseases and arthritis. Aspirin and other salicylatecompounds are frequently used in treatment to interrupt amplification ofthe inflammatory process and temporarily relieve the pain. Other drugcompounds used for these purposes include phenylpropionic acidderivatives such as Ibuprofen and Naproxin, Sulindac, phenyl butazone,corticosteroids, antimalarials such as chloroquine andhydroxychloroquine sulfate, and fenemates. For a thorough review ofvarious drugs utilized in treating rheumatic diseases, reference is madeto J. Hosp. Pharm., 36:622 (May 1979).

Calcium channels are membrane-spanning, multi-subunit proteins thatallow controlled entry of Ca⁺⁺ ions into cells from the extracellularfluid. Such channels are found throughout the animal kingdom, and havebeen identified in bacterial, fungal and plant cells. Commonly, calciumchannels are voltage dependent. In such channels, the “opening” allowsan initial influx of Ca⁺⁺ ions into the cells which lowers the potentialdifference between the inside of the cell bearing the channel and theextracellular medium bathing the cell. The rate of influx of Ca⁺⁺ ionsinto the cell depends on this potential difference. All “excitable”cells in animals, such as neurons of the central nervous system (“CNS”),peripheral nerve cells, and muscle cells, including those of skeletalmuscles, cardiac muscles, and venous and arterial smooth muscles, havevoltage-dependent calcium channels. Calcium channels are physiologicallyimportant because the channels have a central role in regulatingintracellular Ca⁺⁺ ions levels. These levels are important for cellviability and function. Thus, intracellular Ca⁺⁺ ion concentrations areimplicated in a number of vital processes in animals, such asneurotransmitter release, muscle contraction, pacemaker activity, andsecretion of hormones.

It is believed that calcium channels are relevant in certain diseasestates. A number of compounds useful in treating various cardiovasculardiseases in animals, including humans, are thought to exert theirbeneficial effects by modulating functions of voltage-dependent calciumchannels present in cardiac and/or vascular smooth muscle. Many of thesecompounds bind to calcium channels and block, or reduce the rate of,influx of Ca⁺⁺ ions into the cells in response to depolarization of thecell membrane. An understanding of the pharmacology of compounds thatinteract with calcium channels in other organ systems, such as thecentral nervous system, and the ability to rationally design compoundsthat will interact with these specific subtypes of human calciumchannels to have desired therapeutic, e.g., treatment ofneurodegenerative disorders, effects have been hampered by an inabilityto independently determine how many different types of calcium channelsexist or the molecular nature of individual subtypes, particularly inthe CNS, and the unavailability of pure preparations of specific channelsubtypes, i.e., systems to evaluate the specificity of calciumchannel-effecting compounds.

Multiple types of calcium channels have been detected based onelectrophysiological and pharmacological studies of various mammaliancells from various tissues (e.g., skeletal muscle, cardiac muscle, lung,smooth muscle and brain) Bean, B. P., Annu Rev. Physiol. 51:367-384(1989) and Hess, P., Annu. Rev. Neurosci. 56:337 (1990). These differenttypes of calcium channels have been broadly categorized into fourclasses, L-, T-, N-, and P-type, distinguished by current kinetics,holding potential sensitivity and sensitivity to calcium channelagonists and antagonists. Four subtypes of neuronal voltage-dependentcalcium channels have been proposed Swandulla, D. et al., TrendsNeurosci 14:46 (1991). The L-, N- and P-type channels have each beenimplicated in nociception, but only the N-type channel has beenconsistently implicated in acute, persistent and neuropathic pain. Asynthetic version of (ω-conotoxin MVIIA, a 25-amino acid peptide derivedfrom the venom of the piscivorous marine snail, Conus Magus has beenused intrathecally in humans and has ˜85% success rate for the treatmentof pain with a greater potency than morphine.

While known drug therapies have utility, there are drawbacks to theiruse. For instance, it may take up to six months of consistent use ofsome medications in order for the product to have effect in relievingthe patient's pain. Consequently, a particular subject may be receivingtreatment and continuing to suffer for up to six months before thephysician can assess whether the treatment is effective. Many existingdrugs also have substantial adverse side effects in certain patients,and subjects must therefore be carefully monitored. Additionally, mostexisting drugs bring only temporary relief to sufferers and must betaken consistently on a daily or weekly basis for continued relief.Finally, with disease progression, the amount of medication needed toalleviate the pain may increase thus increasing the potential for sideeffects. Thus, there is still a need for an effective and safe treatmentto alleviate pain.

SUMMARY OF THE INVENTION

In one aspect the present invention provides compounds having selectiveaction at N-type calcium channels that are useful for the treatment ofpain.

Compounds of the present invention that show selective action at N-typecalcium channels are compounds in accord with structural diagram I,

wherein,

R¹ is selected from E¹ and E² wherein:

E¹ is N(R⁴)₂ where R⁴ at each occurrence is selected from hydrogen,benzyl, C₁₋₆alkyl, C₃₋₇cycloalkyl, methoxy C₁₋₄alkyl, and

E² is an N-linked heterocyclyl selected from piperidinyl, morpholinyland pyrrolidinyl;

R² is selected from E³ and E⁴, wherein:

E³ is N(R⁵)₂ where R⁵ at each occurrence is independently selected fromhydrogen, C₁₋₆alkyl, phenylC₁₋₆alkyl, C₁₋₆alkenyl, C₃₋₇cycloalkyl,1-methyl-C₃₋₇cycloalkyl, C₁₋₆alkylcarbonyl, C₁₋₆alkoxycarbonyl,tetrahydrofurfuryl, adamantyl, pyridyl, benzthiazolyl, pyrazolyl,1,3-isoindoledion-5-yl, phenyl substituted pyrazolyl, pyrimidinyl andphenyl mono- or di-substituted with a moiety independently selected ateach occurrence from C₁₋₆alkoxy, C₁₋₆alkyl, perhaloC₁₋₆alkyl, halogenand morpholinyl, and E⁴ is selected from nitro, or an N-linkedheterocyclyl selected from piperidinyl, morpholinyl and pyrrolidinyl,and

R³ is selected from C₁₋₆alkyl, phenoxyC₁₋₃alkyl or phenyl substitutedwith E⁵, where E⁵ is selected from hydrogen, halogen, C₁₋₆alkoxy,C₃₋₇cycloalkyl, morpholinyl, C₁₋₄perfluoroalkyl, NHC₁₋₃alkyl andN(C₁₋₃alkyl)₂.

Certain embodiments of the invention are compounds in accord withstructural diagram I wherein R¹ and R³ are as heretofore defined and R²is NHR⁵, where R⁵ is selected from benz[d]thiazol-2-yl,1,5-dimethyl-2-phenyl-1,2-dihydropyrazol-3-on-4-yl,2-phenyl-2,5-dihydro-1H-pyrazol-3-yl, pyrimidin-4-yl,tetrahydrofuran-2-ylmethyl, 2-phenylpropyl and 1-phenylethyl.

Other embodiments of the invention are compounds in accord withstructural diagrams II, III or IV,

wherein R¹, R² and E⁵ are as heretofore defined.

Yet other compounds of the invention are compounds in accord withstructural diagram III where E⁵ is halogen and R¹ and R² are asheretofore defined.

Still other compounds of the invention are compounds in accord withstructural diagram III where E⁵ is fluoro and R¹ and R² are asheretofore defined.

Particular compounds of the invention are compounds in accord withstructural diagram III where R¹ is NHCH₃ or N(CH₃)₂, R² is NHcyclopropylor NHCH₃ and E⁵ is fluoro.

Most particular compounds of the invention are those exemplified herein.

In another aspect, the invention comprises a method for using compoundsaccording to structural diagram I for the treatment of pain, said methodcomprising administering a pain-ameliorating effective amount of anysuch compound.

One embodiment of the method of the invention comprises administering apain-ameliorating effective amount of a compound in accordance withstructural diagram I to a subject in need of treatment for acute,persistent or neuropathic pain.

In a further aspect, the invention comprises methods for makingcompounds in accord with structural diagram I.

In yet another aspect, the invention comprises pharmaceuticalcompositions comprising compounds in accord with structural diagram Itogether with excipients, diluents or stabilisers, as further disclosedherein, useful for the treatment of acute, persistent and neuropathicpain.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of the invention are those within the scope of the genericdescription and particularly those compounds exemplified hereafter.

Suitable pharmaceutically-acceptable salts of compounds of the inventioninclude acid addition salts such as methanesulphonate, fumarate,hydrochloride, hydrobromide, citrate, tris(hydroxymethyl)aminomethane,maleate and salts formed with phosphoric and sulphuric acid.

Where compounds of the present invention possess a chiral center it isto be understood that the invention encompasses all optical isomers anddiastereoisomers of such compounds.

Where compounds of the present invention can tautomerize it is to beunderstood that the invention encompasses all tautomeric forms of suchcompounds.

Where compounds of the present invention can exist in unsolvated as wellas solvated forms such as, for example, hydrated forms, it is to beunderstood that the invention encompasses all such solvated andunsolvated forms.

Another aspect of the invention provides processes for making compoundsof the invention, as follows:

-   a) Preparing novel 3-substituted-3-oxo-propionic acid ethyl esters    (β-keto esters) according to structural diagram V, as follows:    wherein R³ is as heretofore defined;-   b) converting said β-keto esters of structural diagram V to enamines    according to structural diagram VI, as follows    wherein R⁶ is a group selected from —NH—CO—CH₃, NO₂ and Br;-   c) cyclizing said enamines of structural diagram VI to form    compounds according to structural diagram VII, as follows-   d) when R⁶ is —NH—CO—CH₃, converting a compound of structural    diagram VII to a compound according to structural diagram I by the    process of the following scheme:

when R⁶ is —NO₂, converting a compound of structural diagram VII to acompound according to structural diagram I wherein R¹ and R² are bothNH₂, by the process of the following scheme:

or,

when R⁶ is -Br, converting a compound of structural diagram VII to acompound according to structural diagram I wherein R² is E³, by theprocess of the following scheme:

wherein, if necessary, in steps a), b), c) and d) any functional groupis protected with a protecting group, and thereafter,

-   e) removing any said protecting group;-   f) converting one compound according to structural diagram I to    another compound according to structural diagram I by procedures    described in Methods A through L herein, and-   g) purifying said compound of structural diagram I to the extent    necessary and, if necessary, forming a pharmaceutically-acceptable    salt.

To use a compound of the invention or a pharmaceutically-acceptable saltthereof for the therapeutic treatment, which may include prophylactictreatment, of pain in mammals, which may be humans, the compound can beformulated in accordance with standard pharmaceutical practice as apharmaceutical composition. Accordingly, a further aspect of theinvention provides a pharmaceutical composition which contains acompound of the structural diagram I as defined herein or apharmaceutically-acceptable salt thereof, in association with apharmaceutically-acceptable additive such as an excipient or carrier.

Suitable pharmaceutical compositions that contain a compound of theinvention may be administered in conventional ways, for example by oral,topical, parenteral, buccal, nasal, vaginal or rectal administration, orby inhalation. For these purposes a compound of the invention may beformulated by means known in the art in the form of, for example,tablets, capsules, aqueous or oily solutions, suspensions, emulsions,creams, ointments, gels, nasal sprays, suppositories, finely dividedpowders or aerosols for inhalation, and for parenteral use (includingintravenous, intramuscular or infusion) sterile aqueous or oilysolutions or suspensions or sterile emulsions. A preferred route ofadministration is orally by tablet or capsule.

In addition to a compound of the present invention, a pharmaceuticalcomposition of this invention may also contain one or more otherpharmacologically-active agents. Alternatively, a pharmaceuticalcomposition comprising a compound of this invention may beco-administered simultaneously or sequentially with one or more othercompatible pharmacologically-active agents.

Pharmaceutical compositions of this invention will normally beadministered so that a pain-ameliorating effective daily dose isreceived by the subject. The daily dose may be given in divided doses asnecessary, the precise amount of the compound received and the route ofadministration depending on the weight, age and sex of the patient beingtreated and on the particular disease condition being treated accordingto principles known in the art. A preferred dosage regime is once daily.

A yet further embodiment of the invention provide the use of a compoundof the structural diagram I, or a pharmaceutically-acceptable saltthereof, in the manufacture of a medicament useful for binding to N-typecalcium channels in a warm-blooded animal such as a human being.

Still another embodiment of the invention provides a method of binding acompound of the invention to N-type calcium channels of a warm-bloodedanimal, such as a human being, in need of treatment for pain, whichmethod comprises administering to said animal an effective amount of acompound of structural diagram I or a pharmaceutically-acceptable saltthereof.

A further aspect of the present invention provides a pharmaceuticalcomposition which includes a compound of the present invention asdefined herein or a pharmaceutically-acceptable salt thereof, inassociation with a pharmaceutically-acceptable additive such as anexcipient or a carrier.

A still further aspect of the present invention is a method of treatmentof the human or animal body that includes the administration of acompound of the present invention or a pharmaceutically-acceptable saltthereof.

Definitions:

When used herein “halo” or “halogen” means fluoro, chloro, bromo oriodo;

when substituents herein are stated to be “selected from” or“independently selected from” a group of moieties, it is to beunderstood that included compounds are those where all substituents arethe same and compounds where each substituent is different;

when used herein the term “alkyl,” as in for example C₁₋₆alkyl, unlessotherwise defined, includes both straight and branched chain alkylgroups. References to individual alkyl groups such as “propyl” mean thenormal, straight chain form, that is, n-propyl;

when used herein, a term such as “C₁₋₆alkyl” means alkyl groups having1, 2, 3, 4, 5 or 6 carbon atoms and collective groups such as C₁₋₄alkyland includes straight and branched moieties such as methyl, ethyl,iso-propyl and t-butyl, similarly, a term such as “C₁₋₃alkoxy” includesparticular moieties such as methoxy, ethoxy and propoxy, and terms usedherein that are not otherwise defined are intended to have theirconventionally-understood meaning.

The Methods and Examples which follow are intended to illustrate but notlimit the invention. In the Methods and Examples, unless otherwisestated:

concentrations were carried out by rotary evaporation in vacuo;

operations were carried out at ambient temperature, that is in the range18-26° C. and under a nitrogen atmosphere;

column chromatography (by the flash procedure) was performed on MerckKieselgel silica (Art. 9385);

yields are given for illustrative purposes only and are not necessarilythe maximum attainable;

the structure of compounds of the invention were generally confirmed byconventional NMR and mass spectral techniques, peak multiplicities areshown thus: s, singlet; bs, broad singlet; d, doublet; AB or dd, doubletof doublets; t, triplet; dt, double of triplets; m, multiplet; bm, broadmultiplet; FAB m/s data were obtained using a Platform spectrometer(supplied by Micromass) run in electrospray and, where appropriate,either positive ion data or negative ion data were collected, herein(M+H)⁺ is provided;

purity of intermediates were was in general assessed by m/s or NMRanalysis; and where used the following abbreviations have meanings asfollows:

DCM is dichloromethane,

DMF is N,N-dimethylformamide,

DMSO is dimethylsulfoxide,

CDCl₃ is deuterated chloroform,

FAB is fast atom bombardment,

m/s is mass spectroscopy or mass spectral,

NMR is Nuclear Magnetic Resonance,

NMP is N-methylpyrrolidinone, and

THF is tetrahydrofuran.

Biological Methods:

I. N-channel FLIPR (Fluorescent Laser Imaging Plate Reader) Assay.

The methods described herein provide a reliable FLIPR-based readout ofthe efficacy and potency with which test compounds inhibit calcium fluxthrough the N-type calcium channel expressed in its native form in ahuman-derived neuroblastoma cell line differentiated chemically to aneuronal phenotype. The degree to which a compound at a particularconcentration inhibited the N-channel calcium flux was determined bycomparing the amplitude of peak calcium increase in the presence of thecompound to a control 80 mM K⁺ stimulus in wells without compound.Results obtained for this FLIPR assay were validated in two ways:

a) the N-channel specific peptide toxin, conotoxin MVIIA, showed anIC₅₀=3 nM (determined from fit to five-point concentration responseanalysis), compatible with the known literature value; and

b) IC₅₀ values were determined for a set of 18 small molecules fromchemistry lead series (pIC₅₀ range: 4.67-7.02).

Potency of these same test compounds as inhibitors of the N-type calciumcurrent was also determined by direct electrophysiological measurementeither in neuronally differentiated IMR-32 cells, or in freshly-isolatedrat superior cervical ganglion neurons. pIC₅₀'s yielded by the twomethodologies for the compound set were closely comparable (r=0.91;p<0.001).

A. Cell Culture.

An immortalized cell line, IMR32, derived from human neuroblastoma cellsobtained from the ATCC (product #CCL-127) was used for all experiments.Cells were grown in T75 flasks containing Eagle's minimum essentialmedium (MEM) w/Earle's salts and non-essential amino acids withoutglutamine (Cat.#SLM-034-B, Specialty Media, Philipsburg, N.J.), 10% FBSand 1% glutamine. Cells were grown to ˜70-80% confluency (by visualmicroscopic estimation) before sub-culturing. To maintain a stockculture, cultures were split at a ratio of 1:3-1:4 by creating a cellsuspension by trituration, and pipetting a volume of the cell suspensionsufficient to yield this final ratio into new flasks containing ˜20 mLof fresh media. Sub-culturing was generally performed two times perweek. For preparation of 96 well plates (black-walled; Cat # 3603,Costar Co., Cambridge, Mass.), a T75 flask containing cells of desiredconfluency was brought up to 120 mL volume with media. Cells were thenfreed by trituration, and the cell suspension was plated into 12-96 wellplates to yield final well volume of 100 μL.

B. Cell Differentiation to Neuronal Phenotype.

Cells were induced to differentiate in a differentiation mediumconsisting of: MEM, 10% FBS, 1% glutamine, 1 μM 2-butyl-cAMP (49.1mg/100 mL media (Cat. # D-0627, Sigma Corp., St Louis, Mo.), and 2.5 mMbromo-deoxy-uridine (stock: 30.7 mg/10 mL media, 25 mL of abovestock/100 mL media; Sigma Cat .# B-9285). To induce differentiation, thecells were treated with differentiation media (by complete mediumchange) 2 days after an initial plating in 96 well plates. Confluency atthis time was ˜40%. A complete medium change with freshly prepareddifferentiating medium was subsequently performed every 2-3 days. Cellswere exposed to these differentiation conditions for 6 to 11 days beforebeing used in FLIPR experiments.

C. Standard Experimental Solutions.

Solutions of the following composition (in mM) were used in experiments(Buffers without probenicid purchased from Specialty Media (Buffers Aand B: Cat. # BSS053A; Buffers C & D: Cat. # BSS056A).

Buffer A (first wash buffer): Krebs-Ringer-HEPES (KRH) buffer: NaCl:125, KCl: 5, MgSO₄: 1.2, KH₂PO₄: 1.2, CaCl₂ 2H₂O: 2, Glucose: 6, HEPES:25, pH: 7.4 (pH adjusted with NaOH)

Buffer B (dye loading buffer) KRH buffer with 2.5 μM probenicid: same asbuffer A, but probenicid added to final concentration of 2.5 μM.Probenecid (Cat. # P-8761, Sigma Chemical Co., St. Louis, Mo.) made as astock solution at 250 mM.

Buffer C (dye washout buffer) KRH buffer with 0 mM K⁺ and 2.5 μMprobenicid: NaCl: 130, MgSO₄: 1.2, NaH₂PO₄: 1.2, CaCl₂ 2H₂O: 2, Glucose:6, HEPES: 25, pH: 7.4 (pH adjusted with NaOH).

Buffer D (compound dilution buffer): Buffer C with 0.1% w/v bovine serumalbumin (BSA; Sigma).

D. Pharmacological Standards and Compounds.

The following solutions were used to obtain the data disclosed herein.

Nitrendipine: (RBI Chemicals, Natick, Mass.): Stock: 10 mM in DMSO;Pipetting solution: 9 μM; pipette 20 μL into 120 μL volume in well forfinal well concentration: 1 μM.

w-Conotoxin MVIIA: (Cat. # H-8210; Bachem Inc., Torrance, Calif.):Stock: 1 mM in HPLC grade H₂O with 0.1% BSA; Pipetting solution: 4.5 μM;pipette 20 μl into 140 μl volume in well for final well concentration: 1μM.

Test compound stock and solution preparation: Compounds prepared dailyas stocks at 10 mM in 100% DMSO; Pipetting solution: 45 μM or serialdilutions thereof; pipette 20 μL into 140 μL volume in well for finalwell concentration: 1 μM or 10-fold dilutions thereof.

High potassium (depolarization) solution: Buffer C with 240 mM K⁺ added;pipette 80 μL into 160 μL volume in well for final well concentration of80 mM K⁺.

E. Cell Loading with Fluorescent Dyes.

Fluorescent dye solution preparation: A calcium indicator dye, Fluo-4acetylmethylester (Fluo 4-AM; Cat. # F-124201; Molecular Probes, Eugene,OR) was used to measure changes in intracellular free calcium withFLIPR. 1 mM Fluo-4 AM stock solution was made by dissolution in DMSO.This stock was then diluted to 4.6 μM with Buffer B (Fluo-4 AM workingsolution).

Cell loading procedure: Plates containing cells were washed with BufferA using an automated cell washer (Model #: 5161552, Labsystems Oy,Helsinki, Finland) with controls set to the following parameters: cellheight: C/D; cell pulse: 4/5, washes: 3; volume: 5; DRY positionsetting. These settings resulted in a 70 μL residual depth of bufferover cells in each well. 100 μL of the Fluo-4 AM working solution wasthen added to each well resulting in a final Fluo-4 AM concentration of2.7 μM Cells were incubated in this solution at 37° C. for 1-1.5 h.Cells were then washed with Buffer C five times using the cell washerwith parameters the same as the pre-loading washes above with theexceptions of: washes: 5; WET position setting. A final wash was thenconducted by changing the parameters as follows: washes: 1; volume: 2.This resulted in a final well volume of 120 μL. Cells were allowed toequilibrate under this condition for 10 min, and then used in the FLIPRprotocol.

F. FLIPR Protocol

Instrumentation: Real time changes in intracellular free calcium inresponse to potassium-induced depolarization in the absence or presenceof putative N-channel inhibitors were measured by either a FLIPR I orFLIPR II (configured for 96-well format) instrument (Molecular Devices,Sunnyvale, Calif.). Identical settings and protocols were used with eachinstrument, and results obtained from the two instruments wereindistinguishable for a set of standard benchmark compounds.

FLIPR hardware settings: Laser power was set to about 0.3 watts.Excitation wavelength was set to a 488 nm peak, and the emissionwavelength to 540 nm. Camera aperture was set to 2. All experiments wereconducted at room temperature (20-22° C.).

Plate layout—reference signals: Certain wells on each plate wereallocated to standards to determine minimum and maximum specificfluorescent signal against which inhibitory effects of compounds werenormalized. The reference standards were distributed at plate locationsincluding edge and interior wells

Maximum signal (N-channel+non-specific): 12 wells were incubated innitrendipine (1 μM) solution and 80 mM K⁺ added to determine maximalCa²⁺ increase mediated by N-channels+non-specific (non-L-, non-N-channelmediated fluorescence increase). The coefficient of variation amongstthese wells for the K⁺-evoked peak increase in fluorescence units wastypically less than 12%.

Minimum signal (non-specific): 6 wells were incubated in nitrendipine (1μM)+w-conotoxin MVIIA and 80 mM K⁺ added to determine background Ca²⁺with all N-channels pharmacologically occluded. The peak non-specificsignal component was typically less than 15% of the maximum signal peakamplitude.

N-channel reference small molecule: A compound that had beencharacterized extensively with respect to N-channel inhibitory activityin both FLIPR and patch clamp electrophysiology was included on eachplate in triplicate at 1 μM (near IC₅₀) to establish a reference point.

Test compounds: 5 test compounds were evaluated for potency on eachplate. Each compound was tested at 5 increasing concentrations spanninghalf-log units and typically reaching a maximal concentration of 10 μM.Each concentration was tested in triplicate wells.

Protocol structure: The FLIPR protocol was configured as three solutionaddition/sampling sequences (see below). Conotoxin (1 μM final conc.)was added to appropriate wells prior to placing the plate in the FLIPRinstrument. Wells initially contained a total solution volume of 100 μl,and after all three solution additions contained 240 μl. The activemixing (by the pipette) option was not used in any sequence.

Nitrendipine addition sequence: 28 s total duration with fluorescencesignal sampling at 1 Hz for 2 s, followed by addition of 20 μLnitrendipine standard solution at 10 μL/s, followed by sampling at 0.5Hz for 24 s.

Test compound addition sequence: 64 s total duration with sampling at0.5 Hz for 4 sec, test solution addition of 40 μL at 20 μL/s, followedby sampling at 0.2 Hz for 60 s.

Compound incubation, cell depolarization and calcium readout sequence:1024 s total duration with sampling at 0.0167 Hz for 840 s, followed bysolution addition 80 μL of high K⁺ (depolarization) solution, followedby sampling at 1 Hz for 180 sec. This final 180 sec sampling intervalthus represented the epoch where the peak increase in intracellularcalcium due to flux through activated N-channels occurred.

G. Data Analysis

FLIPR software: Prior to export, the data was normalized within theFLIPR software module for two effects.

Baseline correction: The baseline was corrected by “zeroing” at sample #57 (immediately prior to KCl addition). This normalization served tocorrect the y axis offset of the fluorescent trace from each well sothat all traces had a common point just prior to onset of the relevantevoked fluorescent increase.

Spatial uniformity correction factor: The data was normalized by aprocedure which calculates a mean over the plate of fluorescent unitsfrom the first sample, and then multiplies the data from each well by ascalar that adjusts the value of the first sample to this average value,thus normalizing for differences in absolute baseline fluorescenceamongst the wells caused by differences in cell densities or dyeloading.

External software: Data were exported from FLIPR into Excel as “*.squ”extension files. Following export, operations were performed in Excel tocalculate the maximal peak amplitude (relative to the zeroed baseline)of the fluorescence increase following potassium addition in each well.Measurements from wells where an test compound was added were thennormalized as a percentage between the mean amplitudes from thereference wells providing the maximum (100%) and non-specific (0%)signal components, as described above. The resulting percent inhibitionby test compounds was considered to reflect inhibition of calcium fluxat the N-type channel.

II. L-channel FLIPR Assay.

The methods described below provided a reliable FLIPR-based readout ofthe efficacy and potency with which test compounds inhibited calciumflux through the L-type calcium channel expressed natively in ahuman-derived neuroblastoma cell line, SK-N-SH. The degree to which agiven compound concentration inhibited the L-channel was determined bycomparing the amplitude of peak calcium increase to an 80 mM K⁺ stimulusin the test well to the peak increase in wells without compound Theassay was validated by obtaining 5-point concentration-response curvesand thereby determining IC₅₀ values for the reference L-channelblockers, nitrendipine (30 nM), nifedipine and verapamil. These valueswere compatible with the known literature values for these agents toblock Ca²⁺ flux through the L-channel.

A. Cell Culture:

An immortalized cell line, SK-N-SH, derived from human neuroblastomacells (ATCC product # HTB-11) was used for all experiments. Cells weregrown in T75 flasks containing Eagle's minimum essential medium (MEM)w/Earle's salts, with 0.1 mM non-essential amino acids, 1.0 mM Napyruvate and 10% fetal bovine serum (FBS; Cat. # SLM-034-B, SpecialtyMedia). Cells were grown to 100% confluency (by visual microscopicestimation) before sub-culture. Cells were sub-cultured at a ratio of1:3 by first rinsing with 3 mL PBS, replacing the PBS with PBScontaining 0.25% trypsin until the cells detached from the surface. 1 mLof the resulting suspension was then added to a new flask containing 10mL fresh media. Cells were then incubated (37° C., 5% CO₂), and mediawas exchanged about 3 days after subculturing.

B. Preparation of Cells for Experiments:

Cells used for experiments were at the 100% confluency growth stage.Each flask provided enough cells for three 96-well plates. Cells weredetached from the flask by addition of 0.25% trypsin, as described forthe sub-culturing protocol. Once detached, 7 mL fresh media was added tothe flask, and the solution triturated gently. An additional 20 mL mediawas then added, and 100 μL of this final cell suspension was then addedto each well of a 96-well plate. Before use in experiments the plateswere incubated at 37° C. in 5% CO₂ until cells reached 100% confluence(1-2 days).

C. Experimental Procedures:

The composition of solutions, hardware settings, plate layout, structureof the FLIPR protocol, and analytical settings and procedures wereidentical to those described herein for the N-channel assays with thefollowing differences as regards Plate layout and reference signals.

Maximum signal (L-channel+non-specific): 12 wells received 20 μL bufferaddition only (no nitrendipine) in the first solution addition sequenceto define the maximal K⁺-evoked Ca²⁺ increase mediated byL-channels+non-specific (non-L -channel mediated fluorescence increase).The coefficient of variation amongst these wells for the K⁺-evoked peakincrease in fluorescence units was typically less than 12%.

Minimum signal (non-specific): 6 wells were incubated in nitrendipine (1μM), followed by 80 mM K⁺ added to determine background Ca²⁺ with allL-channels pharmacologically occluded. The peak non-specific signalcomponent was typically less than 15% of the maximum signal peakamplitude.

L-channel reference small molecule: Nitrendipine was included intriplicate wells on each plate at 30 nM (near IC₅₀) for a referencereadout.

III. N-channel Patch Clamp Electrophysiology.

Conventional whole cell recording techniques were used to directlymeasure the ability of test compounds to inhibit Ca²⁺ current throughN-type calcium channels. N-type current were recorded from bothneuronally differentiated IMR-32 cells, and native neurons freshlydissociated from superior cervical ganglia of early postnatal rats. Eachday, currents in both cell types were confirmed as N-currents showingthat greater than 90% of the total inward current during depolarizingsteps was blocked by a supramaximal concentration (3 mM) of w-conotoxinMVIIA. Additionally, the potency of w-conotoxin MVIIA was periodicallydetermined to be about 3 nM (IC₅₀), a value consistent with thatreported in the literature. Results for a subset of compounds tested inboth cell types did not differ significantly, thus data are consideredas one data set unless otherwise specified.

A. IMR-32 Cell Culture and Differentiation:

IMR32 cells were cultured and neuronally differentiated using proceduresidentical to those described for the FLIPR N-channel assay except thatfor differentiation cells were plated in 35 mm plexiglass culturedishes, rather than 96-well plates.

B. Dissociation of Rat Superior Cervical Ganglion (SCG) Neurons:

7-10 day old rat pups were euthanized in a chamber containing a high CO₂atmosphere. Immediately, SCG were surgically isolated, removed andplaced in ice cold Hanks balance salt solution (HBSS). SCG's weredesheathed, cut open and placed in a solution of HBSS containing 20 U/mLpapain (37° C.) for 15 min. The papain solution was then exchanged forHBSS (37° C.) containing 16 mg/mL dispase and 400 U/mL collagenase for40 min with gentle trituration of tissue every 15 min. Cells were thenrecovered by centrifugation and stored in L-15 medium at 4° C. for useon the same day. For recording, a drop of cell containing solution wasplaced on a poly-L-lysine coated 35 mm plexiglass culture dish, andcells allowed to adhere for several minutes.

C. Electrophysiological Procedures:

Solutions: Recording solutions were adapted from those described byThompson and Wong (1991) J. Physiol., 439: 671-689. Solutions werestored as aliquots for not more than one month (intracellular, −20° C.,extracellular, 4° C.) before experiments. The pipette (intracellular)solution contained (in mM): TRIS, 130; CsBAPTA, 10; HEPES, 10; Mg²⁺ATP,5; pH to 7.3 with methanesulphonic acid; osmolality ˜315 mOsm.Extracellular solution contained (in mM): TRIS 120; CsCl, 5; HEPES, 10;Mg²⁺Cl, 1; Ba²+Cl, 5, glucose, 25; tetraethylammonium chloride, 15;tetrodotoxin, 200 (added at time of experiment); pH to 7.4 withmethanesulphonic acid; osmolality ˜320 mOsm.

Whole cell recording and analysis: The whole-cell voltage clampconfiguration of the patch clamp technique as described by Hamill et al.(1981) Pflügers Arch. 391: 85-100, was employed to isolatevoltage-dependent calcium currents. Culture dishes containing cells wereplaced in a chamber on the stage of an inverted microscope. Allexperiments were conducted at room temperature (20-22° C.). Patchpipettes were fabricated from thin-wall glass (1.5 mm OD, 1.12 mm ID;World Precision Instruments, New Haven, Conn.) on the Brown-Flaming P-86puller (DC resistance: 3-6 MΩ; Sutter Instr. Co., Novato, Calif.). AnAxopatch 1B amplifier (Axon Instruments, Foster City, Calif.) was usedto obtain current signals and this was connected to a personal computerby either a TL-1 (Scientific Solutions, Solon, Ohio) or Digidata 1200(Axon Instr.) interface. The current signal was balanced to zero withthe pipette immersed in the bath just prior to forming a seal on theneuron. Seal resistance ranged from 1 to greater than 10 GΩ. Seriesresistance was usually less than 10 MΩ, and was not compensatedelectronically. Digitized data acquisition and voltage step protocolswere accomplished with pClamp 6.0 software (Axon Instr). Data werelow-pass filtered at less than one-half the digital sampling rate priorto digitizing. To record N-type currents for evaluation of inhibitorypotency of compounds (steady-state concentration-response analysis), 200ms voltage steps to +10 mV were delivered at 15 sec intervals from aholding potential of −90 mV. The recorded currents were leak subtractedon-line with a P-4 or P-6 subpulse protocol in the pClamp software. Toevaluate open channel block of compounds, 10 ms voltage steps to +10 mVwere delivered at varying frequencies from a holding potential of −90 mVwithout using on-line leak subtraction. These voltage protocols bothyielded constant inward current amplitudes over 5-10 minutes ofrecording. Peak current amplitude was analyzed using the clampfit moduleof pClamp software. Origin 5.0 software (Microcal Corp, Northampton,Mass.) was used to iteratively fit concentration-response data to astandard Hill function, and to provide graphic displays for currenttraces and analyzed data.

Drug/compound preparation and delivery: Test compounds were prepared as10 mM stock solutions in DMSO, and appropriate volumes of these stocksolutions dissolved into extracellular buffer to yield the desiredconcentrations. Solutions containing drugs/compounds were appliedfocally from any of six linearly arranged glass-lined tubes (200 mmo.d., Hewlett Packard, Wilmington, Del.) positioned ˜100 mm from therecorded neuron. Each solution was released from the desired tube by anelectronically controlled solenoid valve system (BME Systems, Baltimore,Md.). This system achieved rapid (<100 ms) equilibration of drugsolution in the extracellular phase without perturbing the recordingcharacteristics.

Compounds of the invention generally had a binding affinity, expressedas the IC₅₀ (μM), for the N-type calcium channel, as measured by theFLIPR assay, of about 10 μM of less. Table 1 shows the results forcertain compounds of the invention. TABLE 1 Example No. IC₅₀ (μM) 136.28 3 17.23 4 3.54 5 5.83 6 6.11 7 1.99 9 2.71 10 3.81 11 6.91 12 1.8913 10.48 14 2.59 15 4.13 16 81.58 19 7.96 20 3.18 21 2.54 23 3.51 242.68 25 4.83 26 4.90 27 5.55 28 3.81 29 5.44 32 6.46 33 2.82 33 2.82 372.18 38 9.15 39 4.53 40 2.98 41 1.80 44 5.39 48 7.74 49 5.88 50 5.90 513.78 53 7.19 54 1.75 55 2.31 56 6.38IV. Formalin Test.

The formalin test assesses the inhibitory effects of orally administeredN-type calcium channel antagonists on formalin-induced nocifensivebehaviours in rats. The formalin test is a well established pain test(Dubuisson and Dennis, 1977; Wheeler-Aceto et al., 1990; Coderre et al.,1993). This test consists of two distinct phases of formalin-inducedbehaviour. The first phase response, occurring between 0 to 5 minutes,is caused by acute nociception to the noxious chemical (formalin)injected into the paw. This is followed by a quiescent period of between5 to 15 min post injection. A second phase response, occurring after 15minutes and lasting up to 60 minutes, is caused by sensitisation of thecentral neurons in the dorsal horn. Central sensitisation augments thenoxious afferent input and a stronger pain barrage is transmitted intothe brain. Inhibition of the second phase response is indicative of acentral mechanism of drug action.

The procedure for the formalin test is as follows: male rats are placedin a plexiglass chamber and observed for 30-45 min. to observe theirbaseline activity. Multiple groups of animals are pretreated with eithervehicle or different doses of a test compound. Animals are dosed withthe drug of interest either 40 min., if by the intraperitoneal route, or90 min., if by the oral route, prior to injection of formalin into ahind paw (under the dorsal skin; 0.05 mL of sterile 5% formalin). Thenumber of paw flinches and licks during first phase (0-5 min.) andsecond phase (20-35 min.) are scored and recorded. Flinch and lickresponses are calculated as percentage of inhibition compared with themean score of a saline control group. Drug potencies are expressed asthe dose which causes 50% of the maximum inhibitory effect (“ID₅₀”).Student t-tests are used for statistical analysis to determine thesignificance of drug effects. Compounds are considered active based ontheir ability to inhibit the flinch response.

V. Chronic Constrictive Injury Test.

The Chronic Constrictive Injury (“CCI”) test or Neuropathic Pain Modelassesses neuropathic pain associated with nerve injuries that can arisedirectly from trauma and compression, or indirectly from diseasesranging from infection to cancer, metabolic conditions, toxins,nutritional deficiencies, immunological dysfunction and musculoskeletalchanges. In the CCI model (Bennett and Xie, 1988) a unilateralperipheral neuropathy is produced in rats by partial nerve ligation.

Sprague-Dawley rats (250-350 g) are anesthetized with sodiumpentobarbital and the common sciatic nerve is exposed at the level ofthe mid thigh by blunt dissection through the biceps femoris. A sectionof nerve (about 7 mm), proximal to the sciatic trifurcation, is exposedand ligated 4 times with chromic gut suture. The suture is tied withabout 1 mm spacing between ligatures. The incision is closed in layersand the animals are allowed to recover. Thermal hyperalgesia is measuredusing the paw-withdrawal test (Hargreaves et al, 1988). Nervecompression due to the partial nerve ligation causes shorter latenciesfor paw withdrawal compared to the latency of paw withdrawal of paws ofnormal or sham operated legs. Animals are habituated on an elevatedglass floor. A radiant heat source is aimed at the mid-plantar hindpaw(sciatic nerve territory) through the glass floor with a 20 secondcut-off used to prevent injury to the skin. Latencies for the withdrawalreflex in both paws are recorded. Response to test compounds areevaluated at different times following oral administration to determineonset and duration of drug effect. Dose response studies are conductedwith multiple groups of CCI rats dosed orally with either vehicle or thetest compound for 5 days. Paw withdrawal latencies are measured each dayprior to the first daily dose. Data analysis is performed by multiplemeans comparison (Dunnett's test) and drug potencies are expressed asthe dose which causes 50% of the maximum efficacy (“EC₅₀”).

Chemical Methods:

Method A:

Exemplary compound 19, 2-(4-cyclohexylphenyl)-quinoline-4,6-diamine, seeTable 1 hereafter, was made by the following method. Other compoundsprepared by Method A from suitable precursors are listed in Table 1.

A1. 3-(4-Cyclohexylphenyl)-3-oxo-propionic acid ethyl ester:

Into a three-neck 2 L round-bottom flask equipped with an additionfunnel, nitrogen inlet, magnetic stirrer, heating mantle, thermocoupleand condenser, was placed 21.7 g (0.543 moles) of a 60%-in-oildispersion of sodium hydride. To this was added dry hexane (1 L). Theresulting suspension was stirred for 15 minutes, stirring was halted andthe solids were allowed to settle. The clear supernatant containing thehexane and dissolved oil was then removed via a cannula. Diethylcarbonate(1 L) was added and the suspension was heated to 120° C. Tothis suspension was cautiously added dropwise, over 40 minutes, asolution of 100 g (0.494 moles) of 4′-cyclohexyl actephenone dissolvedin 250 mL of diethyl carbonate. As addition proceeded a reactioninitiated, hydrogen was evolved and the color changed to tan. After theacetophenone derivative addition was complete, the reaction was heatedfor 1 additional hour. The reaction mixture was cooled and was pouredinto a 2 L separatory funnel. The diethyl carbonate layer was twicewashed with 10% acetic acid solution and then dried over MgSO₄. Thesolution was then filtered and concentrated on a rotary evaporatorfollowed by pumping with high vacuum at 70° C. for 18 hr. Theconcentrated solution crystallised on cooling over 24 hrs to give acolorless solid. The product obtained was then used without furtherpurification, yield 133 g (98%). 1H NMR reveals that the β-keto esterproduct actually exists as a keto-enol tautomer mixture in solution,with the keto form predominant in the solid.

A2. 3-(4-Acetylamino-phenylamino)-3-(4-cyclohexylphenyl)-acrylic acidbutyl ester:

Into a 1 liter single-neck round-bottom flask equipped with a Soxhletextractor apparatus with condenser, magnetic stirrer and nitrogen inletwas placed 50.25 g (0.183 moles) of3-(4-cyclohexylphenyl)-3-oxo-propionic acid ethyl ester, 25 g (0.167moles) of 4′-aminoacetani 1.55 g (0.008 moles) 4′-aminoacetanilidehydrochloride salt and 500 mL of dry n-butanol. Into the Soxhlet thimble(33×118 mm) was placed highly activated 4A sieves (1.7-2.4 mm beads).These sieves are activated immediately before use under high vacuum withheating (400° C. for 30 min). The mixture was then brought to refluxsuch that the butanol azeotropically removed water, driving theequilibrium reaction, and the water was removed from the butanol by thesieves before being returned to the reaction pot. The reaction wasallowed to continue for 48 hrs. It was necessary to replace the chargeof sieves after the first 24 hrs. Transesterification to the butyl esteralong with removal of ethanol occurs concomitantly with enamineformation. After 48 hrs the reaction pot was cooled, then placed in a−40° C. freezer and crystals were allowed to form over 24 hrs. Thecrystals were collected by vacuum filtration and the solids washed withcold ethanol. The product was then dried in a vacuum oven to give 73.8 g(98%) of the desired enamine.

A3. N-[2-(4-Cyclohexylphenyl)-4-hydroxy-quinolin-6-yl]-acetamide:

Into a 2 L three-neck round-bottom flask equipped with a condenser,magnetic stirrer, thermocouple, heating mantle with a variable voltagecontroller, and a nitrogen inlet, was charged 1.2 L of Dowtherm A (aeutectic mixture of 26.5% diphenyl and 73.5% diphenyl oxide). TheDowtherm A was then preheated to 250° C. To this was cautiously added insmall portions 48 g (0.11 moles) of3-(4-acetylamino-phenylamino)-3-(4-cyclohexylphenyl)-acrylic acid butylester. As portions were added gas was evolved and foaming occurred.Crystals of product begin to form and adhere to the sides. After all thematerial had been added the heating of the reaction was continued for 1hour. The mixture was then cooled to room temperature and hexane wasadded. The solid product was collected by vacuum filtration and washedwith hexane. After drying in a vacuum oven, 35.7 g (90%) of product wasrecovered.

A4. N-[2-(4-Cyclohexylphenyl)-4-methoxy-quinolin-6-yl]-acetamide:

Into a 500 mL three-neck round-bottom flask equipped with a condenser,magnetic stirrer and nitrogen inlet was added 35.7 g (0.099 moles) ofN-[2-(4-cyclohexylphenyl)-4-hydroxy-quinolin-6-yl]-acetamide. Thematerial was suspended in 250 mL of toluene with stirring and then 20.6mL (0.21 moles) of dimethyl sulfate was added. The resulting suspensionwas heated with a silicone oil heating bath, to gentle a reflux for 18hr. After this time the reaction was allowed to cool and hexane wasadded. The solids were collected by vacuum filtration and washed withhexane. After drying, the solids were suspended in a 2 L Erlenmeyerflask with 1 L of 5% sodium hydroxide solution. The vigorously stirredsuspension was then heated to 70° C. for 30 min. This step converted thesalt form of the material to the free base and removed some impurities.After cooling the solids were collected by vacuum filtration and thenwashed with water. The product was dried in a vacuum oven to give 35.8 g(96%) of a material which contained approximately 30% of side products,with the N-methylated material constituting the major impurity. Thismaterial was used in the following procedure without furtherpurification.

A5. N-[4-Amino-2-(4-cyclohexylphenyl)-quinolin-6-yl]-acetamide:

Into a 500 mL three-neck round-bottom flask equipped with a mechanicalstirrer and condenser, nitrogen inlet and gas outlet was placed 35 g ofN-[2-(4-cyclohexylphenyl)-4-methoxy-quinolin-6-yl]-acetamide and 250 gof ammonium acetate. The stirred solid suspension was then brought to upto 115° C. Ammonia evolution began, and the material slowly fused anddissolved in the acetic acid that formed over time. The temperature wasslowly raised to 140° C. over 1 hr. Caution was used to ensure that thecondenser and gas outlet remained clear of solid ammonium acetate, whichcan collect on cool surfaces from sublimation of excess ammoniumacetate. After 4 hours of heating, the reaction was cooled and pouredinto 1 L of water. The pH was then adjusted to 9.5 by the slow additionof concentrated a NaOH solution with application of ice cooling. Ethylacetate was then added and the mixture was filtered. Solid impurities,some of which are due to N-methylated side products from the previousstep, are removed and the liquid filtrate was poured into a 2 Lseparatory funnel. The ethyl acetate layer was separated, washed twicewith 5% NaOH solution and then dried over Na₂SO₄. After filtration, thesolvent was evaporated to give 22 g (60%) of a solid which mostlyconsisted of the product and about 20% of the 6-N-deacetylated material,2-(4-cyclohexylphenyl)-4,6-quinolinediamine. This product was then usedwithout further purification in the following acetate-removal step.

A6. 2-(4-Cyclohexylphenyl)-4,6-quinolinediamine:

The 22 g of material from the previous step was placed in a 1 L flask towhich 500 mL of 6 N HCl was added. The mixture was then heated withstirring to 95° C. for 18 hrs. After this time, the solution was cooledin ice and was then cautiously neutralised with concentrated NaOHsolution, followed by adjustment to pH 9.5. The solution was then pouredinto a 2 L separatory funnel and extracted with ethyl acetate. Theorganic layer was then dried over Na₂SO₄, filtered and concentrated togive 17 g of product. Repeated crystallisation using methanol, methylenechloride and hexane gave 9.0 g (41%) of the diamino quinoline product.

A7: 3-(Methyl-phenoxy)-3-oxo-propionic acid ethyl ester:

3-(Methyl-phenoxy)-3-oxo-propionic acid ethyl ester used for thepreparation of the compound of Example 14, was prepared as follows. In a12 L round bottom flask equipped with mechanical stirrer, thermometer,addition funnel and nitrogen inlet was placed 160.0 grams (60%, 4.0moles) of sodium hydride dispersion in mineral oil. The flask was cooledwith an ice bath and 3.5 L of dry THF added with stirring, whilemaintaining the temperature below 20° C. To the suspension of stirredsodium hydride was added 376.4 grams (4.0 moles) of phenol dissolved in0.3 L of THF. The addition was carried out at such a rate so as tomaintain the temperature below 10° C. over the course of approximately 2hours. The mixture was then allowed to warm to room temperature and stirfor 1 hour. To the re-cooled solution, over the course of 1 hour, wasadded 418 grams (2.0 moles) of ethyl 4-bromoacetoacetate (A. Svendsenand P. M. Boll, Tetrahedron 1973 29, 4251-4258) dissolved in 0.3 L ofTHF. The rate of addition was controlled so as to maintain thetemperature below 10° C. The ice bath was removed and the brown slurrystirred overnight at room temperature. The reaction was quenched bypouring into 2.2 L of 1.0 N hydrochloric acid and the phases separated.The aqueous phase was extracted with 0.5 L of diethyl ether; thecombined organic phase washed with 1.0 L of saturated brine and driedover MgSO₄. After filtering and removal of solvent a red-brown oil wasobtained, 830 gram of crude product. The crude product was dissolved in0.4 L of hexane and applied to a column of 8.0 L of silica wet-packed inhexane. The column was eluted with 4.0 L of hexane; 8.0 L of 3:1 hexaneto diethyl ether and 12.0 L of 2:1 hexane to diethyl ether. The secondfraction, 459.3 gram, was reapplied to a column of 4.0 L of silicawet-packed in hexane. The column was eluted with 3.0 L of 95:5 hexane todiethyl ether; 2.0 L of 9:1 hexane to diethyl ether; 2.0 L of 4:1 hexaneto diethyl ether and 12.0 L of 3:1 hexane to diethyl ether. The majorfraction was bulb-to-bulb distilled using a Kugelrohr apparatus and anoven temperature of 40-45° C. at <1.0 torr. The desired product wasobtained as an oil, 145.9 gram (33%).

Method B:

Exemplary compound 10, 2-(3-fluorophenyl)-quinoline-4,6-diamine, seeTable 1 hereafter, was made by the following method. Other compoundsprepared by Method B from suitable precursors are listed in Table 1.

B1. 3-(3-Fluorophenyl)-3-oxo-propionic acid ethyl ester:

The title compound was prepared from m-fluoro acetophenone, by a methodanalogous to the preparation of 3-(4-cyclohexylphenyl)-3-oxo-propionicacid ethyl ester in step A1 of Method A, except that the product waspurified by vacuum distillation (bp 114-117° C. at 0.8-0.9 mm Hg) in 91%yield.

B2. 3-(4-Nitro-phenylamino)-3-(3-fluorophenyl)-acrylic acid butyl ester:

Into a dry 2 L round-bottom flask was placed 106.3 grams (0.506 moles)of 3-(3-fluorophenyl)-3-oxo-propionic acid ethyl ester, 63.0 grams(0.456 moles) of 4-nitro-aniline and 4.0 grams (0.023 moles) of4-nitro-aniline hydrochloride. To the mixture was added 1.3 L ofn-butanol, the flask was fitted with a Soxhlet extractor (cup volume of0.3 L), condenser and nitrogen inlet. Dry, activated 4 Å sieves (200grams) were placed in the extractor cup and the reaction mixture heatedto reflux temperature of 118° C. under nitrogen and maintained at thattemperature for 90 hours. The reaction mixture was decanted while hotfrom a small amount of solids and chilled to −15° C. for 48 hoursCrystalline solids were collected by vacuum filtration. The crystalswere washed with 0.2 L of cold ethanol and two 0.2 L portions of hexanesand vacuum dried at 50° C. overnight to yield 35.8 grams (20.8% yield)of the title compound.

The mother liquors were concentrated, diluted with 1.0 L of toluene andthe toluene removed in vacuo; this process was repeated two times. Theliquors were then diluted in 1.0 L of n-butanol and another 1.75 grams(10.0 mmol) of 4-nitro-aniline hydrochloride added; the flask was fittedwith a soxhlet extractor as before and the cup charged with a fresh 200grams of sieves. The mixture was placed under nitrogen and brought toreflux temperature for 90 hours. The reaction was cooled and thenreduced to a final volume of 0.6 L in vacuo. The solution was thenseeded with crystalline3-(4-nitro-phenylamino)-3-(3-fluorophenyl)-acrylic acid butyl ester andlet stand at −15° C. for 48 hours. The crystals were collected asbefore; 64.3 grams obtained after drying. Analysis showed this materialto be contaminated with 4-nitro-aniline and it was purified by flashchromatography. The product was dissolved in 0.5 L of 1:1 methylenechloride to hexane and applied to a column of 3.0 L of silica wet-packedin 1:1 methylene chloride to hexane. The column was eluted with 4.0 L of1: 1 methylene chloride to hexane; 9.0 L of 2:1 methylene chloride tohexane; and 2.0 L of methylene chloride. Fractions of 0.5 L werecollected and those containing the desired product combined to yield41.7 grams (24.3%)of bright yellow solid. The combined yield was 45.1%.

B3. 2-(3-Fluorophenyl)-6-nitro-quinolin-4-ol:

In a 3 L three-neck flask, equipped with mechanical stirrer, Claisenadapter holding a thermocouple probe and reflux condenser with nitrogeninlet was added 0.75 L of Dowtherm A (a eutectic mixture of 26.5%diphenyl and 73.5% diphenyl oxide) The solvent is then preheated to 250°C. To this was cautiously added in small portions 77.0 grams (0.215moles) of 3-(4-nitro-phenylamino)-3-(3-fluorophenyl)-acrylic acid butylester over the course of 0.25 hours. The mixture was maintained at 250°C. for 1.5 hours and then allowed to cool to 90° C. over the course of 2hours. The mixture was treated with 1.0 L of hexanes, and allowed tocool to room temperature while stirring overnight. The tan solids werecollected by suction filtration and washed with three 0.15 L portions ofhexanes. The solids were dried under vacuum at 50° C. overnight to yield58.63 grams (96.0%) of the title compound.

B4. 6-Bromo-4-chloro-2-(3-fluorophenyl)-quinoline:

Into a 500 mL three-neck round-bottom flask equipped with a condenser,magnetic stirrer and nitrogen inlet was placed 5.2 g (16.3 mmoles) of6-bromo-2-(3-fluorophenyl)-quinolin-4-ol. To this was added 15.2 mL(25.0 g, 163 mmoles, 10 equiv.) of phosphorus oxychloride with stirring.The mixture was then heated to 110° C. for 4 hr. At the end of this timethe reaction was cooled to room temperature and water cautiously addeddropwise until all of the POCl₃ was consumed. The product crystallisedfrom the water and the solids were collected by filtration. The solidswere washed with water and placed in a 250 mL Erlenmeyer where they werethen triturated with water. After collection by filtration, washing withwater, and drying in a vacuum oven, 4.6 g (84%) of the pure product wasobtained.

B5. 6-Bromo-4-azido-2-(3-fluorophenyl)-quinoline:

Into a 250 mL three-neck round-bottom flask equipped with a condenser,magnetic stirrer, silicone oil heating bath, nitrogen inlet and gasoutlet was placed 3.2 g (9.50 mmoles) of6-bromo-4-chloro-2-(3-fluorophenyl)-quinoline. To this was added 75 mLof N-methyl pyrrolidinone then 6.0 g (95 mmoles, 10 equiv.) of sodiumazide. The stirring mixture was then warmed to 60° C. for 18 hr. At theend of this time the reaction was cooled, then poured into a 1 Lseparatory funnel containing 500 mL water and 250 mL of ethyl acetate.The pH was adjusted to 9.0 the layers were separated. The aqueous layerwas then extracted twice with 100 mL of ethyl acetate. The organiclayers were combined, dried over Na₂SO₄, filtered and concentrated togive a product, which was carried to the next step without furtherpurification.

B6. 4-Azido-2-(3-fluorophenyl)-6-nitro-quinoline:

The title compound was prepared by a procedure analogous to that of stepB5 for 6-bromo-4-azido-2-(3-fluorophenyl)-quinoline. The product wasisolated on a 16.16 mmole scale.

B7. 2-(3-Fluorophenyl)-4,6-quinolinediamine:

Into a 500 mL three-neck flask equipped with a condenser, magneticstirrer, nitrogen inlet gas outlet and a silicone oil heating bath wasplaced 4-azido-2-(3-fluorophenyl)-6-nitro-quinoline. The material wassuspended in 250 mL of ethyl acetate and 50 mL of ethanol. The stirredmixture was heated to reflux, then 20 g (89 mmoles, 6 equiv.) ofstannous chloride dihydrate was cautiously added portionwise over 40min. The reaction was then heated for an additional 2 hr. At the end ofthis time, the reaction was cooled and then poured into 500 mL of water.The pH was cautiously adjusted to 9.0 and the solution filtered. Thesolids were washed with 100 mL of ethyl acetate, the filtrates combinedand the aqueous layer extracted twice with 200 mL of ethyl acetate. Theorganic layers were combined, dried over Na₂SO₄, filtered andconcentrated. The product was chromatographed on a silica gel column,using 10% methanol in ethyl acetate as eluent, and was thenrecrystallised from methylene chloride and hexane to give 3.6 g (88%) ofthe product.

Method C:

Exemplary compound 29, N6-cyclopropyl-2-(3-fluorophenyl)-N4,N4-dimethyl-quinoline-4,6-diamine, see Table 1 hereafter, was made bythe following method. Other compounds prepared by Method C from suitableprecursors are listed in Table 1.

C1. 3-(3-Fluorophenyl)-3-oxo-propionic acid ethyl ester:

Preparation of the title compound is described in step B 1 of Method B.

C2. 3-(4-Bromo-phenylamino)-3-(3-fluorophenyl)-acrylic acid butyl ester:

The title compound was prepared in 86% yield by a procedure analogous tothe preparation of3-(4-acetylamino-phenylamino)-3-(4-cyclohexylphenyl)-acrylic acid butylester, step A2 of Method A.

C3. 6-Bromo-2-(3-fluorophenyl)-quinolin-4-ol:

The title compound was prepared in a 92% yield by a procedure analogousto the preparation of 2-(3-fluorophenyl)-6-nitro-quinolin-4-ol, step B3of Method B.

C4. 6-Bromo-4-chloro-2-(3-fluorophenyl)-quinoline:

Into a 500 mL three-neck round-bottom flask equipped with a condenser,magnetic stirrer and nitrogen inlet was placed 5.2 g (16.3 mmoles) of6-bromo-2-(3-fluorophenyl)-quinolin-4-ol. To this was added 15.2 mL(25.0 g, 163 mmoles, 10 equiv.) of phosphorus oxychloride with stirring.The mixture was heated to 110° C. for 4 hr. At the end of this time thereaction was cooled to room temperature and water was cautiously addeddropwise until all of the POCl₃ was consumed. The product crystallisedfrom the water and solids were collected by filtration. The solids werewashed with water, placed in a 250 mL Erlenmeyer and triturated withwater. After collection by filtration, washing with water, and drying ina vacuum oven, 4.6 g (84%) of the product was obtained.

C5(a). N-[6-bromo-2-(3-fluorophenyl)-4-quinolinyl]-N4, N4-dimethylamine:

Into a 500 mL three-neck round-bottom flask equipped with magneticstirrer, nitrogen inlet, gas outlet, condenser and heating bath wasplaced 20 g (59.4 mmoles of6-bromo-4-chloro-2-(3-fluorophenyl)-quinoline. The material wasdissolved in 150 mL of N-methyl pyrrolidinone and 250 mL of a 40% aq.solution of dimethylamine was added to the stirring mixture. Thereaction was then warmed to 60° C. for 48 hrs. At the end of this timethe reaction was cooled, into 3 L of water in a 4 L Erlenmeyer flask andthe mixture was stirred until solids formed. The solids were collectedby vacuum filtration and dried in a vacuum oven. The product wasrecrystallised from ethanol in a −20° C. freezer to give 19.6 g (95%)yield of the aminated product.

C5(b). Alternative procedure:

N-[6-bromo-2-(3-fluorophenyl)-4-quinolinyl]-N4, N4-dimethylamine wasalternatively prepared as follows. Into a 1 L Parr bomb equipped withmechanical stirring, thermocouple, heater with controller and pressuregauge was placed 20 g (59.4 mmoles of6-bromo-4-chloro-2-(3-fluorophenyl)-quinoline. To this was added 350 mLof ethanol and 350 mL of a 40% aq. solution of dimethylamine. The bombwas sealed and the stirred mixture was then heated to At the end of thistime the reaction was allowed to cool to room temperature and was thenvented. The contents were then poured into 3 L of water in a 4 LErlenmeyer flask and the mixture was stirred until solids formed. Thesolids were collected by vacuum filtration and then dried in a vacuumoven. The crude product was recrystallised from ethanol in a −20° C.freezer to give 18.15 g (92%) yield of the aminated product.

C6. N6-Cyclopropyl-2-(3-fluorophenyl)-N4,N4-dimethyl-quinoline-4,6-diamine:

The title compound was prepared in a fashion analogous to thepreparation of N6-cyclopropyl-2-(3-chlorophenyl)-N4,N4-dimethyl-quinoline-4,6-diamine (Method D2).

Method D:

Exemplary compound 55,N6-cyclopropyl-N4,N4-dimethyl-2-(3-morpholin-4-yl-phenyl)-quinoline-4,6-diamine,see Table 1 hereafter, was made by the following method. Other compoundsprepared by Method D from suitable precursors are listed in Table 1.

D1. N-[6-Bromo-2-(3-chlorophenyl)-4-quinolinyl]-N4,N4-dimethylamine:

The title compound was prepared in a manner analogous to the procedureof step C5a or C5b of Method C.

D2.2-(3-Chlorophenyl)-N6-cyclopropyl-N4,N4-dimethyl-quinoline-4,6-diamine:

The title compound, Example 53, see Table 1 hereafter, was prepared byselective Pd catalyzed substitution of a 6-bromo moiety in the presenceof a m-chloro substituent, as follows.

Into each of two 4 dram vials equipped with a magnetic stir bar and ateflon lined septum closure, was placed 1.18 g (3.27 mmoles, 1 equiv.)of N-[6-bromo-2-(3-chlorophenyl)-4-quinolinyl]-N,N-dimethylamine, 73 mgof tris(dibenzylideneacetone) dipalladium(0) (0.08 mmoles, 0.025 equiv.,0.05 equiv. Pd), 397 mg of racemic2,2′-bis(diphenylphosphino)-1,1′-bisnaphthyl (0.64 mmoles, 0.2 equiv.),943 mg of sodium tert-butoxide (9.81 mmoles, 3 equiv.) and, finally, 10mL of THF and of 2 mL of cyclopropylamine (28.9 mmoles, 8.8 equiv.). Thevial was placed under a nitrogen atmosphere, sealed and then heated to70° C. in an oil bath for 18 hr. At the end of this time the reactionvials were cooled, combined, and poured into a 1 L separatory funnelcontaining 300 mL of ethyl acetate and 500 mL of 1 N NaOH solution. Thelayers were separated and the organic layer was washed again with 500 mLof 1 N NaOH solution. The organic layers were then dried over Na₂SO₄,filtered and concentrated. The residue was then purified by flashchromatography using 5% ethyl acetate in methylene chloride containing0.5% triethylamine. Fractions containing the desired compound were thencombined. The combined fractions were concentrated and the resultingresidue crystallised with methylene chloride and hexane to give 1.22 g(55%) of pure product. A later fraction containing 273 mg (14% yield )was also recovered, which corresponded to the decyclopropylatedmaterial, N-[2-(3-chlorophenyl)-6-amino-4-quinolinyl]-N-dimethylamine.

D3.N6-Cyclopropyl-N4,N4-dimethyl-2-[3-(4-morpholinyl)phenyl]-4,6-quinolinediamine:

The title compound was prepared by Pd catalyzed substitution of am-chloro moiety. Into each of two 4-dram vials equipped with a magneticstir bar and a teflon-lined septum closure, was placed 1.18 g (3.27mmoles, 1 equiv.) ofN-[6-cyclopropyl-2-(3-chlorophenyl)-4-quinolinyl]-N,N-dimethylamine, 73mg of tris(dibenzylideneacetone) dipalladium(0) (0.08 mmoles, 0.025equiv., 0.05 equiv. Pd), 148 mg of 2-(di-t-butylphosphino)biphenyl (0.49mmoles, 0.15 equiv.), 943 mg of sodium tert-butoxide (9.81 mmoles, 3equiv.) and, finally, 10 mL of THF and of 1 mL of morpholine(11.46mmoles, 3.5 equiv.). The vial was placed under a nitrogen atmosphere,sealed and then heated to 70° C. in an oil bath for 18 hr. At the end ofthis time the reaction vials are cooled, combined, and poured into a 1 Lseparatory funnel containing 300 mL of ethyl acetate and 500 mL of 1 NNaOH solution. The layers are separated and the organic layer was washedagain with 500 mL of 1 N NaOH solution. The organic layers are thendried over Na₂SO₄, filtered and concentrated. The residue was thenpurified by flash chromatography using 40% ethyl acetate in methylenechloride containing 0.5% triethylamine. Fractions containing the desiredcompound were then combined. Fractions were concentrated and theresulting residue crystallised with methylene chloride and hexane togive 1.51 g (59%) of product.

Method E:

Exemplary compound 56,2-(3-morpholin-4-yl-phenyl)-N4,N4-dimethyl-quinoline-4,6-diamine, seeTable 1 hereafter, was made by the following method. Other compoundsprepared by Method E from suitable precursors are listed in Table 1.

E1.N6-Cyclopropyl-N4,N4-dimethyl-2-[3-(4-morpholinyl)phenyl]-4,6-quinolinediamine:

The title compound was prepared according to the procedure of step D3 ofMethod D.

E2. N4,N4-Dimethyl-2-[3-(4-morpholinyl)phenyl]-4,6-quinolinediamine:

The title compound was prepared by acid catalyzed removal of acyclopropyl group, as follows. Into a 100 mL round-bottom flask equippedwith a condenser, magnetic stirrer, nitrogen inlet and silicone oilheating bath, was placed 600 mg ofN6-cyclopropyl-N4,N4-dimethyl-2-[3-(4-morpholinyl)phenyl]-4,6-quinolinediamine.To this was added 30 mL of tretrahydrofuran, 15 mL of water and then 30mL of conc. HCl solution. The mixture was heated to 65° C. for 3 days.At the end of this time the starting material had disappeared asdetermined by TLC. The reaction was then cooled, the pH adjusted to 9.0with sodium hydroxide solution and poured into a 1 L separatory funnel,where it was extracted with 300 mL of ethyl acetate. The organic layerwashed with 1 N NaOH, dried over Na₂SO₄, filtered and concentrated. Theresidue was subjected to flash chromatography using 5% methanol in a50:50 ethyl acetate and methylene chloride mixture containing 0.5%triethylamine to give 305 mg (57%) of the product.

Method F:

Exemplary compound 91,2-(3-fluorophenyl)-N4,N4-dimethyl-N6-cyclopropyl-N6-acetyl-quinoline-4,6-diamine,see Table 1 hereafter, was made by the following method. Other compoundsprepared by Method F from suitable precursors are listed in Table 1.

F1.N6-Cyclopropyl-N4,N4-dimethyl-2-[3-(4-morpholinyl)phenyl]-4,6-quinolinediamine:

The title compound was prepared according to the procedure of step D3 ofMethod D.

F2.N-Cyclopropyl-N-[4-(dimethylamino)-2-(3-fluorophenyl)-6-quinolinyl]acetamide:

The title compound was prepared by acetylation at the 6-nitrogen asfollows. Into a 50 mL round-bottom flask equipped with a magneticstirrer, condenser silicone oil bath and nitrogen inlet, was placed 750mg (2.33 mmoles) ofN6-cyclopropyl-N4,N4-dimethyl-2-[3-fluorophenyl]-4,6-quinolinediamine.To this was added 5 mL of glacial acetic acid. After the materialdissolved, 202 mg (2.57 mmoles, 1.1 equiv.) of acetyl chloride wasadded. The mixture was stirred at room temperature for 1 hr, then heatedto 60° C. for 2 hr. At the end of this time the reaction was cooled andpoured into a 1 L separatory funnel containing 300 mL ethyl acetate and500 mL of 1 N NaOH solution. The organic layer was separated, washedwith NaOH solution, dried over Na₂SO₄, filtered and concentrated. Theresidue was then crystallised from methylene chloride and hexane to give550 mg (65%) of product.

Method G:

Exemplary compound 18,2-(3-methoxyphenyl)-N6-methyl-quinoline-4,6-diamine, see Table 1hereafter, was made by the following method. Other compounds prepared byMethod G from suitable precursors are listed in Table 1.

G1. 2-(3-methoxyphenyl)-4,6-quinolinediamine:

The title compound was prepared in a manner analogous to the procedureof Method A.

G2. N-[4-Amino-2-(3-methoxyphenyl)-quinolin-6-yl]-formamide:

Formic anhydride was prepared by placing 260 mg (5.66 mmoles) of formicacid into a dry 100 mL round-bottom flask equipped with a condenser,magnetic stirrer and a nitrogen inlet. The flask was cooled with an icebath and then 470 mg (4.61 mmoles) of acetic anhydride was added. Thestirring solution was then gently warmed to 50° C. with an oil bath for2 hrs. After this time the reaction was cooled, and 25 mL of dry THF wasadded followed by the addition of 1.0 g (3.77 mmoles) of2-(3-methoxyphenyl)-4,6-quinolinediamine as a solution in 10 mL of dryTHF. The mixture was then heated to reflux for 2 hrs. At the end of thistime the reaction was cooled and ether was added. The solids whichformed were then collected by filtration and washed with ether. Thesolids were then dissolved in ethyl acetate and 5% NaOH solution, thelayers separated and the organic layer dried over Na₂SO₄, filtered andconcentrated to give 830 mg (75%) of the desired product. This productwas used without further purification.

G3. 2-(3-Methoxyphenyl)-N-6-methyl-4,6-quinolinediamine:

Into a 200 mL round-bottom flask equipped with a condenser, magneticstirrer and nitrogen inlet, was placed 830 mg (2.83 mmoles) ofN-[4-amino-2-(3-methoxyphenyl)-quinolin-6-yl]-formamide. To this wasadded 40 mL of dry THF followed by the cautious addition of 40 mL of a1.0 Molar solution of Borane THF complex. The solution was then heatedto reflux for 18 hrs. At the end of this time the reaction was cooled inice and then cautiously quenched by the slow addition of 10 mL drymethanol followed by the cautious slow addition of 40 mL of a 1.0 Msolution of ethereal hydrogen chloride. After allowing the reaction toproceed for 1 hr, the reaction was poured into mixture of ethyl acetateand water and then the pH was adjusted to 9.5. the layers were separatedand the organic layer was dried over Na₂SO₄, filtered and concentrated.The resulting product was then recrystallised from CH₂Cl₂ and hexane togive 720 mg (91%) of the pure 6-N-methyl quinoline.

Method H:

Exemplary compound 27,2-(3-fluorophenyl)-N4,N4-dimethyl-quinoline-4,6-diamine, see Table 1hereafter, was made by the following method. Other compounds prepared byMethod H from suitable precursors are listed in Table 1.

H1. 2-(3-Fluorophenyl)-6-nitro-quinolin-4-ol:

Preparation of the title compound is described in Method B.

H2. N-[2-(3-Fluorophenyl)-6-nitro-4-quinolinyl]-N,N-dimethylamine:

The title compound was prepared in a manner analogous to the preparationof N-[6-bromo-2-(3-fluorophenyl)-4-quinolinyl]-N,N-dimethylamine asdescribed in step C5 of Method C.

H3(a). N-[2-(3-Fluorophenyl)-6-amino-4-quinolinyl]-N,N-dimethylamine:

The title compound was prepared by reduction of a nitro group bycatalytic hydrogenation as follows. Into a 500 mL Parr shaker bottle wasplaced 4.0 g of theN-[2-(3-fluorophenyl)-6-nitro-4-quinolinyl]-N,N-dimethylamine along with150 mg of a catalyst consisting of 5% palladium on calcium carbonatesupport. To this was added 150 mL of ethanol, followed by application ofa 50 psi hydrogen atmosphere. The reaction was shaken for 18 hr then thehydrogen atmosphere was replaced by nitrogen. The catalyst was removedby filtration, and the solution concentrated. The residue was taken upin ethyl acetate, washed with 5% sodium hydroxide solution, then theorganic layer was dried over Na₂SO₄, filtered and concentrated. Theextract was recrystallised from methylene chloride and hexane to give2.8 g (77%) of the product.

H3(b). Alternative procedure:

Alternatively,N-[2-(3-fluorophenyl)-6-amino-4-quinolinyl]-N,N-dimethylaminewas prepared in a manner analogous to the preparation of2-(3-fluorophenyl)-4,6-quinolinediamine by reduction of the 6-nitrogroup using stannous chloride as described in step B6 of Method B.

Method I:

Exemplary compound 37,2-(3-fluorophenyl)-N4,N6-dimethyl-quinoline-4,6-diamine, see Table 1hereafter, was made by the following method. Other compounds prepared byMethod I from suitable precursors are listed in Table 1.

I1. 6-Bromo-4-chloro-2-(3-fluorophenyl)-quinoline:

The title compound was prepared as described in step C4 of Method C.

I2. 2-(3-Fluorophenyl)-N4,N6-dimethyl-4,6-quinolinediamine:

The title compound was prepared by simultaneous Pd catalyzedsubstitution of 4-chloro and 6-bromo.

Into each of two 4 dram vials equipped with a magnetic stir bar and ateflon-lined septum closure, was placed 1.1 g (3.27 mmoles, 1 equiv.) of6-bromo-4-chloro-2-(3-fluorophenyl)-quinoline, 73 mg oftris(dibenzylideneacetone) dipalladium(0) (0.08 mmoles, 0.025 equiv.,0.05 equiv. Pd), 397 mg of racemic2,2′-bis(diphenylphosphino)-1,1′-binapthyl (0.64 mmoles, 0.2 equiv.),943 mg of sodium tert-butoxide (9.81 mmoles, 3 equiv) and, finally, 10mL of a 2.0 M solution of methylamine in THF (20 mmoles, 6 equiv). Thevials were placed under a nitrogen atmosphere, sealed and then heated to70° C. in an oil bath for 18 hr. At the end of this time the vials werecooled, combined, and poured into a 1 L separatory funnel containing 300mL of ethyl acetate and 500 mL of 1 N NaOH solution. The layers wereseparated and the organic layer was washed again with 500 mL of 1 N NaOHsolution. The organic layers were then dried over Na₂SO₄, filtered andconcentrated. The residue was purified by flash chromatography using 5%ethyl acetate in methylene chloride containing 0.5% triethylamine. Thefractions containing the desired compound were combined and concentratedand the resulting residue crystallised from methlyene chloride andhexane to give 1.2 g (61%) of product.

Method J:

Exemplary compound 61,2-(3-dimethylaminophenyl)-N4,N4-dimethyl-quinoline-4,6-diamine, seeTable 1 hereafter, was made by the following method. Other compoundsprepared by Method J from suitable precursors are listed in Table 1.

J1. N-[2-(3-Chlorophenyl)-6-nitro-4-quinolinyl]-N,N-dimethylamine:

The title compound was prepared in a manner analogous to the preparationof N-[2-(3-fluorophenyl)-6-nitro-4-quinolinyl]-N,N-dimethylamine asdescribed in step H2 of Method H.

J2. 2-(3-Dimethylaminophenyl)-N4,N4-dimethyl-quinoline-4,6-diamine:

The title compound was prepared fromN-[2-(3-chlorophenyl)-6-nitro-4-quinolinyl]-N,N-dimethylamine by theprocedure of step D2 of Method D using dimethylamine.

Method K:

Exemplary compound 81,2-(3-chlorophenyl)-N6-tert-butylcarbamoyl-quinoline-4,6-diamine,M376003, see Table 1 hereafter, was made by the following method. Othercompounds prepared by Method K from suitable precursors are listed inTable 1.

K1. [4-Amino-2-(3-chlorophenyl)-quinolin-6-yl]-carbamic acid tert-butylester (6-N-BOC protected):

Into a 250 mL three-neck round-bottom flask equipped with a magneticstirrer, condenser, addition funnel, heating bath and nitrogen inlet,was added 1.5 g (5.58 mmoles, 1 equiv.) of2-(3-chlorophenyl)-4,6-quinolinediamine, 50 ml of dry THF, and 3.9 mL(27.9 mmoles, 5 equiv.) of dry triethylamine. To this stirring mixturewas then added over 30 min., 6.7 mL (6.69 mmoles, 1.2 equiv.) ofdi-t-butyl dicarbonate as a solution in 50 mL of THF. The mixture wasthen heated to reflux for 18 hr., and, at the end of this time, cooled,cautiously quenched with water and partitioned between 300 mL of ethylacetate and 500 mL of 5% NaOH solution. The organic layer was washedwith NaOH solution, dried over Na₂SO₄, filtered and concentrated. Theresidue was subjected to flash chromatography, using an eluent of 25%ethyl acetate in methylene chloride containing 0.5% triethylamine togive 1.lg (53%) of the product.

Method L:

Exemplary compound 25, 2-(3-chlorophenyl)-quinoline-4,6-diamine, seeTable 1 hereafter, was made by the following method. Other compoundsprepared by Method L from suitable precursors are listed in Table 1.

L1. 2-(3-Chlorophenyl)-4,6-quinolinediamine:

The BOC protecting group was removed by the following procedure. Into a200 mL round bottom flask equipped with a magnetic stir bar and nitrogeninlet was placed 1.0 g (2.71 mmoles, 1 equiv) of[4-amino-2-(3-chlorophenyl)-quinolin-6-yl]-carbamic acid tert-butylester and 60 mL of a 1:2 mixture of trifluoroacetic acid and methylenechloride. The mixture was stirred at room temperature for 3 hrs., thenconcentrated. The residue was made basic with dilute NaOH solution thenextracted with ethyl acetate. The organic layer was washed with NaHOsolution, dried over Na₂SO₄, filtered and concentrated. The residue wascrystallized from methylene chloride and hexanes to give 700 mg (95%) ofthe title compound.

Exemplary Compounds

Exemplary compounds 1 to 111 inclusive are disclosed in Table 2 whichshows the name of each compound and the method of preparation. Where themethod of preparation is described as C* compounds were prepared by amethod analogous to that of method C, using methylamine in place ofdimethylamine. TABLE 2 Method Ex. No. Name of Preparation 12-(4-Chlorophenyl)-quinoline-4,6-diamine A 22,N6-Dimethyl-quinoline-4,6-diamine G 3 2-Phenyl-quinoline-4,6-diamine A4 2-Phenyl-N4-benzyl-quinoline-4,6-diamine H 52-(3-Trifluoromethylphenyl)-quinoline-4,6-diamine A 62-Phenyl-N4,N4-dimethyl-quinoline-4,6-diamine H 72-Phenyl-N4-propyl-quinoline-4,6-diamine H 82-(4-Fluorophenyl)-quinoline-4,6-diamine A 92-(3-Methoxyphenyl)-quinoline-4,6-diamine A 102-(3-Fluorophenyl)-quinoline-4,6-diamine A & B 112-(4-Methoxyphenyl)-quinoline-4,6-diamine A 122-Pentyl-quinoline-4,6-diamine A 132-(2-Methoxyphenyl)-quinoline-4,6-diamine A 142-Phenoxymethyl-quinoline-4,6-diamine A 152-(2-Chlorophenyl)-quinoline-4,6-diamine A 162-(2-Fluorophenyl)-quinoline-4,6-diamine A 172-Phenyl-N6-methyl-quinoline-4,6-diamine G 182-(3-Methoxyphenyl)-N6-methyl-quinoline-4,6-diamine G 192-(4-Cyclohexylphenyl)-quinoline-4,6-diamine A 202-(4-Bromophenyl)-quinoline-4,6-diamine A 212-(3-Bromophenyl)-quinoline-4,6-diamine A 222-(4-Chlorophenyl)-N6-methyl-quinoline-4,6-diamine G 232-(2-Fluorophenyl)-N6-methyl-quinoline-4,6-diamine G 242-(3-Fluorophenyl)-N6-methyl-quinoline-4,6-diamine G 252-(3-Chlorophenyl)-quinoline-4,6-diamine A & L 262-(3-Fluorophenyl)-N4,N4,N6,N6-tetramethyl-quinoline-4,6- C diamine 272-(3-Fluorophenyl)-N4,N4-dimethyl-quinoline-4,6-diamine H 282-(3-Fluorophenyl)-N4,N4,N6-trimethyl-quinoline-4,6- C diamine 292-(3-Fluorophenyl)-N4,N4-dimethyl-N6-cyclopropyl- Cquinoline-4,6-diamine 302-(3-fluorophenyl)-N4,N4-dimethyl-N6-adamantan-1-yl- Cquinoline-4,6-diamine 312-(3-fluorophenyl)-N4,N4-dimethyl-N6-ethyl-quinoline-4,6- C diamine 322-(3-Fluorophenyl)-N4-morpholin-4-yl-N6-cyclopropyl- Cquinoline-4,6-diamine 33 2-(3-Fluorophenyl)-N4,N4-dimethyl-N6-isopropyl-C quinoline-4,6-diamine 342-(3-Fluorophenyl)-N4-pyrrolidin-1-yl-N6-cyclopropyl- Cquinoline-6-amine 35 2-(3-Fluorophenyl)-N4,N4-dimethyl-N6-cycloheptyl- Cquinoline-4,6-diamine 362-(3-fluorophenyl)-N4-piperidin-1-yl-N6-cyclopropyl- Cquinolin-4,6-diamine 372-(3-Fluorophenyl)-N4,N6-dimethyl-quinoline-4,6-diamine I & C 382-(3-Fluorophenyl)-N4,N4-dimethyl-6-pyrrolidin-1-yl- C quinolin-4-amine39 2-(3-Fluorophenyl)-N4-methyl-quinoline-4,6-diamine H 402-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(6-amino-pyridin-2- Cyl)-quinoline-4,6-diamine 412-(3-Fluorophenyl)-N4-methyl-N6-cyclopropyl-quinoline- C* 4,6-diamine 422-(2-Fluorophenyl)-N4,N4-dimethyl-N6-cyclopropyl- Cquinoline-4,6-diamine 432-(3-Fluorophenyl)-N4,N4-dimethyl-N6-benzothiazol-2-yl- Cquinoline-4,6-diamine 442-(3-Fluorophenyl)-N4,N4-dimethyl-N6-4-(1,5-dimethyl- C2phenyl-1,2-dihydro-pyrazol-3-one)quinoline-4,6-diamine 452-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(2-phenyl-2,5- Cdihydro-1H-pyrazol-3-yl)-quinoline-4,6-diamine 46[2-(3-Fluorophenyl)-N4,N4-dimethyl-N6-piperidin-1-yl- Cquinoline-4,6-diamine 472-(3-Fluorophenyl)-N4,N4-dimethyl-N6-pyrimidin-4-yl- Cquinoline-4,6-diamine 482-(3-Fluorophenyl)-N4,N4-dimethyl-N6-cyclobutyl- C quinoline-4,6-diamine49 2-(3-Fluorophenyl)-N4,N4-dimethyl-N6-cyclopropylmethyl- Cquinoline-4,6-diamine 502-(3-Fluorophenyl)-N4,N4-dimethyl-N6-cyclopentyl- Cquinoline-4,6-diamine 512-(3-Fluorophenyl)-N4,N4-dimethyl-N6-allyl-quinoline-4,6- C diamine 522-(3-Fluorophenyl)-N4,N4-dimethyl-N6-propyl-quinoline- C 4,6-diamine 532-(3-Chlorophenyl)-N4,N4-dimethyl-N6-cyclopropyl- Cquinoline-4,6-diamine 542-(3-Fluorophenyl)-N4-(2-methoxy-ethyl)-N6-cyclopropyl- Cquinoline-4,6-diamine 55 2-(3-Morpholin-4-ylphenyl)-N4,N4-dimethyl-N6- Dcyclopropyl-quinoline-4,6-diamine 562-(3-Morpholin-4-ylphenyl)-N4,N4-dimethyl-quinoline-4,6- E diamine 57[2-(3-Fluorophenyl)-N4,N4-dimethyl-6-nitro-quinoline-4- H amine 58[2-(3-Chlorophenyl)-N4,N4-dimethyl-6-nitro-quinoline-4- H amine 592-(3-Chlorophenyl)-N4,N4-dimethyl-quinoline-4,6-diamine H 602-(3-Fluorophenyl)-N4-methyl-N6-cyclobutyl-quinoline-4,6- C* diamine 612-(3-Dimethylaminophenyl)-N4,N4-dimethyl-quinoline-4,6- J diamine 622-(3-Methylaminophenyl)-N4-methyl-quinoline-4,6-diamine J 632-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(2-methoxy-ethyl)- Cquinoline-4,6-diamine 642-(3-Fluorophenyl)-N4-ethyl-N6-cyclopropyl-quinoline-4,6- C* diamine 652-(3-Methylaminophenyl)-N4,N4-dimethyl-N6-cyclopropyl- Dquinoline-4,6-diamine 662-(3-Fluorophenyl)-N4,N6-dicyclopropyl-quinoline-4,6- I diamine 672-(3-Fluorophenyl)-N4-cyclopropyl-N6-methyl-quinoline- C* 4,6-diamine 682-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(tetrahydro-furan-2- Cylmethyl)-quinoline-4,6-diamine 692-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(2-phenyl-propyl)- Cquinoline-4,6-diamine 702-(3-fluorophenyl)-N4,N4,N6-trimethyl-N6-Ethyl-quinoline- C 4,6-diamine71 2-(3-Fluorophenyl)-N4,N4-dimethyl-N6-((S)-1-phenyl- Cethyl)-quinoline-4,6-diamine 722-(3-Fluorophenyl)-N4-Ethyl-N6-methyl-quinoline-4,6- C diamine 732-(3-Chlorophenyl)-N4,N6-dimethyl-quinoline-4,6-diamine I 742-(3-Fluorophenyl)-N4-(2-methoxy-ethyl)-N6-methyl- C*quinoline-4,6-diamine 752-(3-Chlorophenyl)-N4,N4,N6-trimethyl-quinoline-4,6- C diamine 762-(3-Morpholin-4-yl-phenyl)-N4,N6-Dimethyl-quinoline- D 4,6-diamine 772-(4-Methoxyphenyl)-N6-acetamido-quinoline-4,6-diamine A 782-(4-Bromophenyl)-N6-tert-butylcarbamoyl-quinoline-4,6- K diamine 792-(2-Fluorophenyl)-N6-acetamido-quinoline-4,6-diamine F 802-(3-Bromophenyl)-N6-tert-butylcarbamoyl-quinoline-4,6- K diamine 812-(3-Chlorophenyl)-N6-tert-butylcarbamoyl-quinoline-4,6- K diamine 822-(2-Chlorophenyl)-N6-tert-butylcarbamoyl-quinoline-4,6- K diamine 832-(3-Fluorophenyl)-N6-tert-butylcarbamoyl-quinoline-4,6- K diamine 842-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(4-methoxyphenyl)- Cquinoline-4,6-diamine 85 2-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(3- Caminophenyl)quinoline-4,6-diamine 862-(3-Fluorophenyl)-N4,N4-dimethyl-quinoline-4-amine-6- H & F acetamide87 2-[3-(4-Morpholinyl)phenyl]-N4,N4,N6-trimethyl-quinoline- C & J4,6-diamine 88 2-[3-(Methylamino)phenyl]-N4,N6-dimethyl-quinoline-4,6-C* & J diamine 89 2-(3-Chlorophenyl)-N4-methyl-N6-cyclopropyl-quinoline-C* 4,6-diamine 90 2-[3-(4-Morpholinyl)phenyl]-N4-methyl-N6-cyclopropyl-C* & J quinoline-4,6-diamine 912-(3-Fluorophenyl)-N4,N4-dimethyl-N6-cyclopropyl-N6- C & Facetyl-quinoline-4,6-diamine 922-(3-Fluorophenyl)-N4-methyl-N6-cyclopropyl-N6-acetyl- C* & Fquinoline-4,6-diamine 932-(3-Fluorophenyl)-N4,N4-dimethyl-N6-methyl-N6-acetyl- I & Fquinoline-4,6-diamine 94N6-Cyclopentyl-2-(3-fluorophenyl)-N4-methyl-4,6- C* quinolinediamine 95N6,2-bis(3-fluorophenyl)-N4,N4-dimethyl-4,6- C quinolinediamine 96N6-(3-chloro-4-methylphenyl)-2-(3-fluorophenyl)-N4,N4- Cdimethyl-4,6-quinolinediamine 972-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(2-methyl-1H- Cisoindole-1,3(2H)-dione-5-yl)-quinoline-4,6-diamine 982-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(2,6- Cdichlorophenyl)-quinoline-4,6-diamine 992-(3-Fluorophenyl)-N4-methyl-N6-acetyl-quinoline-4,6- H & F diamine 1002-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(2-chloro-6- Cmethylphenyl)-quinoline-4,6-diamine 1012-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(3,5- Cdimethoxyphenyl)-quinoline-4,6-diamine 1022-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(3,4- Cdichlorophenyl)-quinoline-4,6-diamine 1032-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(3-fluoro-4- Cmethylphenyl)-quinoline-4,6-diamine 1042-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(4-methylphenyl)- Cquinoline-4,6-diamine 1052-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(4-chlorophenyl)- Cquinoline-4,6-diamine 1062-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(3-ethylphenyl)- Cquinoline-4,6-diamine 1072-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(1-methyl- Ccyclopropyl)-quinoline-4,6-diamine 1082-(3-Fluorophenyl)-N4-methyl-N6-(1-methyl-cyclopropyl)- C*quinoline-4,6-diamine 1092-(3-Fluorophenyl)-N4,N4-dimethyl-N6-(1-methyl- Ccyclobutyl)-quinoline-4,6-diamine 1102-(3-Fluorophenyl)-N4-methyl-N6-(1-methyl-cyclobutyl)- C*quinoline-4,6-diamine 1112-(3-Fluorophenyl)-N4-methyl-N6-(iso-propyl)-quinoline- C* 4,6-diamine

Exemplary compounds 1 to 111 inclusive are illustrated in Table 3 whichshows the mass spectroscopy data and the elemental analysis determinedfor certain compounds. TABLE 3 Ex. No. Formula M + 1 Anal. Calc. forTheor. Found 1 C₁₅H₁₂ClN₃ 270/ C₁₅H₁₂ClN₃ C, 65.98; C, 66.00; 272(+)0.05CH₂Cl₂ H, 4.45; H, 4.69; N, 15.34 N, 15.00 2 C₁₁H₁₃N₃ 188(+)C₁₁H₁₃N₃ C, 69.41; C, 69.73; 0.046CH₂Cl₂ H, 6.90; H, 6.95; N, 21.98 N,21.60 3 C₁₅H₁₃N₃ 236(+) C₁₅H₁₃N₃ C, 75.90; C, 75.46; 0.03CH₂Cl₂ H, 5.36;H, 5.74; N, 17.67 N, 17.37 4 C₂₂H₁₉N₃ 326(+) C₂₂H₁₉N₃ C, 79.17; C,78.48; 0.12CH₂Cl₂ H, 5.78; H, 5.86; N, 12.52 N, 12.51 5 C₁₆H₁₂F₃N₃304(+) C₁₆H₁₂F₃N₃ C, 62.42; C, 62.58; 0.12CH₂Cl₂ H, 3.96; H, 4.11; N,13.58 N, 13.28 6 C₁₇H₁₇N₃ 264(+) C₁₇H₁₇N₃ C, 76.23; C, 76.34; 0.25H₂O H,6.59; H, 6.57; N, 15.69 N, 15.35 7 C₁₈H₁₉N₃ 278(+) C₁₈H₁₉N₃ C, 76.95; C,76.66; 0.20H₂O H, 6.96; H, 6.96; N, 14.96 N, 14.96 8 C₁₅H₁₂FN₃ 254(+)C₁₅H₁₂FN₃ C, 69.62; C, 69.78; 0.20H₂O H, 5.01; H, 5.03; 0.10C₄H₈0₂ N,15.81 N, 15.55 9 C₁₆H₁₅N₃O 266(+) C₁₆H₁₅N₃O C, 71.95; C, 71.86; 0.10H₂OH, 5.74; H, 5.68; N, 15.73 N, 15.89 10 C₁₅H₁₂FN₃ 254(+) C₁₅H₁₂FN₃ C,70.38; C, 69.65; 0.15H₂O H, 4.84; H, 4.77; N, 16.42 N, 16.65 11C₁₆H₁₅N₃O 266(+) C₁₆H₁₅N₃O C, 70.98; C, 71.10; 0.08CH₂Cl₂ H, 5.62; H,5.60; N, 15.44 N, 15.62 12 C₁₄H₁₉N₃ 230(+) C₁₄H₁₉N₃ C, 73.32; C, 73.51;H, 8.35; H, 8.32; N, 18.32 N, 18.07 13 C₁₆H₁₅N₃O 266(+) C₁₆H₁₅N₃O C,71.16; C, 71.40; 0.07CH₂Cl₂ H, 5.63; H, 5.84; N, 15.49 N, 15.18 14C₁₆H₁₅N₃O 266(+) C₁₆H₁₅N₃O C, 70.62; C, 70.58; 0.10CH₂Cl₂ H, 5.60; H,5.60; N, 15.34 N, 15.11 15 C₁₅H₁₂ClN₃ 270/ C₁₅H₁₂ClN₃ C, 65.70; C,65.45; 272(+) 0.30C₄H₈0₂ H, 4.90; H, 4.93; N, 14.19 N, 14.52 16C₁₅H₁₂FN₃ 254(+) C₁₅H₁₂FN₃ C, 71.13; C, 71.00; H, 4.78; H, 4.98; N,16.59 N, 16.39 17 C₁₆H₁₅N₃ 250(+) C₁₆H₁₅N₃ C, 75.29; C, 75.09;0.10CH₂Cl₂ H, 6.28; H, 6.29; 0.10C₆H₁₄ N, 15.77 N, 15.42 18 C₁₇H₁₇N₃O280(+) C₁₇H₁₇N₃O C, 69.72; C, 69.67; 0.20CH₂Cl₂ H, 5.92; H, 6.17; N,14.18 N, 13.92 19 C₂₁H₂₃N₃ 318(+) C₂₁H₂₃N₃ C, 78.23; C, 78.08; 0.30CH₄0H, 7.46; H, 7.21; N, 12.85 N, 12.96 20 C₁₅H₁₂BrN₃ 314/ C₁₅H₁₂BrN₃ C,56.92; C, 56.93; 316(+) 0.10CH₂Cl₂ H, 4.14; H, 4.11; 0.10C₆H₁₄ N, 12.68N, 12.47 21 C₁₅H₁₂BrN₃ 314/ C₁₅H₁₂BrN₃ C, 55.66; C, 55.49; 316(+)0.15CH₂Cl₂ H, 3.79; H, 4.07; N, 12.85 N, 13.02 22 C₁₆H₁₄ClN₃ 284/C₁₆H₁₄ClN₃ C, 65.49; C, 65.34; 286(+) 0.20CH₂Cl₂ H, 5.30; H, 5.28;0.15C₆H₁₄ N, 13.40 N, 13.53 23 C₁₆H₁₄FN₃ 268(+) C₁₆H₁₄FN₃ C, 70.61; C,70.91; 0.27H₂O H, 5.39; H, 5.57; N, 15.43 N, 15.04 24 C₁₆H₁₄FN₃ 268(+)C₁₆H₁₄FN₃ C, 69.00; C, 69.04; 0.22CH₂Cl₂ H, 5.73; H, 5.45; 0.20C₆H₁₄ N,13.85 N, 13.89 25 C₁₅H₁₂ClN₃ 270/ C₁₅H₁₂ClN₃ C, 66.79; C, 66.78; 272(+)H, 4.48; H, 4.51; N, 15.58 N, 15.27 26 C₁₉H₂₀FN₃ 310(+) C₁₉H₂₀FN₃ C,59.69; C, 59.51; 2.00HCl H, 5.80; H, 6.04; N, 10.99 N, 10.65 27C₁₇H₁₆FN₃ 282(+) C₁₇H₁₆FN₃ C, 72.57; C, 72.64; H, 5.73; H, 5.74; N,14.94 N, 14.82 28 C₁₈H₁₈FN₃ 296(+) C₁₈H₁₈FN₃ C, 57.30; C, 57.44; 2.00HClH, 5.61; H, 5.51; N, 11.13 N, 11.04 29 C₂₀H₂₀FN₃ 322(+) C₂₀H₂₀FN₃ C,74.74; C, 74.41; H, 6.27; H, 6.26; N, 13.07 N, 12.73 30 C₂₇H₃₀FN₃ 416(+)C₂₇H₃₀FN₃ C, 77.96; C, 78.20; 0.02CH₂Cl₂ H, 7.53; H, 7.16; 0.15C₆H₁₄ N,9.77 N, 9.40 31 C₁₉H₂₀FN₃ 310(+) C₁₉H₂₀FN₃ C, 73.43; C, 73.18;0.04CH₂Cl₂ H, 6.49; H, 6.49; N, 13.57 N, 13.18 32 C₂₂H₂₂FN₃O 364(+)C₂₂H₂₂FN₃O C, 72.71; C, 72.50; H, 6.10; H, 6.10; N, 11.56 N, 11.83 33C₂₀H₂₂FN₃O 324(+) C₂₀H₂₂FN₃O C, 55.50; C, 55.79; 3.00HCl H, 5.82; H,5.69; N, 9.70 N, 9.41 34 C₂₂H₂₂FN₃O 348(+) C₂₂H₂₂FN₃O C, 74.69; C,74.71; 0.10CH₂Cl₂ H, 6.41; H, 6.28; 0.05C₆H₁₄ N, 11.67 N, 11.31 35C₂₄H₂₈FN₃ 378(+) 36 C₂₃H₂₄FN₃ 362(+) C₂₃H₂₄FN₃ C, 74.99; C, 74.95;0.10CH₂Cl₂ H, 6.59; H, 6.67; N, 11.34 N, 11.17 37 C₁₇H₁₆FN₃ 282(+)C₁₇H₁₆FN₃ C, 72.58; C, 72.67; H, 5.73; H, 5.73; N, 14.94 N, 14.85 38C₂₁H₂₂FN₃ 336(+) C₂₁H₂₂FN₃ C, 70.50; C; 70.25; H, 4.89; H, 5.10; N,10.72 N, 10.54 39 C₁₆H₁₄FN₃ 268(+) C₁₆H₁₄FN₃ C, 71.21; C, 71.49; 0.16H₂OH, 5.41; H, 5.45; 0.02C₆H₁₄ N, 15.45 N, 15.09 40 C₂₂H₂₀FN₅ 374(+)C₂₂H₂₀FN₅ C, 70.08; C, 70.23; 0.20H₂O H, 5.45; H, 5.47; N, 18.56 N,18.51 41 C₁₉H₁₈FN₃ 308(+) C₁₉H₁₈FN₃ C, 72.62; C, 72.91; 0.10CH₂Cl₂ H,5.81; H, 5.85; N, 13.30 N, 13.40 42 C₂₀H₂₀FN₃ 322(+) C₂₀H₂₀FN₃ C, 74.71;C, 74.91; 0.16H₂O H, 6.71; H, 6.32; 0.10C₆H₁₄ N, 12.47 N, 12.09 43C₂₄H₁₉FN₄S 44 C₂₈H₂₆FN₅O 466(−) 45 C₂₆H₂₂FN₅ 424(+) 46 C₂₂H₂₄FN₃ 350(+)C₂₂H₂₄FN₃ C, 61.51; C, 61.61; 2.00HCl H, 6.29; H, 6.22; 0.40H₂O N, 9.78N, 9.77 47 C₂₁H₁₈FN₅ 360(+) C₂₁H₁₈FN₅ C, 70.01; C, 70.22; 0.05CH₂Cl₂ H,5.41; H, 5.05; 0.15C₆H₁₄ N, 18.60 N, 18.62 48 C₂₁H₂₂FN₃ 336(+) C₂₁H₂₂FN₃C, 76.30; C, 76.77; 0.10C₆H₁₄ H, 5.75; H, 5.76; N, 12.35 N, 11.97 49C₂₁H₂₂FN₃ 336(+) C₂₁H₂₂FN₃ C, 75.30; C, 75.31; 0.05C₆H₁₄ H, 6.73; H,6.68; N, 12.37 N, 11.99 50 C₂₂H₂₄FN₃ 350(+) C₂₂H₂₄FN₃ C, 75.81; C,76.12; 0.10C₆H₁₄ H, 7.15; H, 6.96; N, 11.73 N, 11.53 51 C₂₀H₂₀FN₃ 322(+)C₂₀H₂₀FN₃ C, 74.74; C, 74.94; H, 6.27; H, 6.30; N, 13.07 N, 12.71 52C₂₀H₂₂FN₃ 324(+) C₂₀H₂₂FN₃ C, 73.63; C, 73.76; 0.05CH₂Cl₂ H, 6.92; H,6.77; 0.05C₆H₁₄ N, 12.65 N, 12.28 53 C₂₀H₂₀ClN₃ 338/340(+) C₂₀H₂₀ClN₃ C,70.86; C, 71.06; 0.015CH₂Cl₂ H, 5.95; H, 5.99; N, 12.39 N, 12.22 54C₂₁H₂₂FN₃O 352(+) C₂₁H₂₂FN₃O C, 71.36; C, 71.31; 0.03CH₂Cl₂ H, 6.28; H,6.26; N, 11.87 N, 12.15 55 C₂₄H₂₈N₄O 389(+) C₂₄H₂₈N₄O C, 74.32; C,74.59; 0.03CH₂Cl₂ H, 7.71; H, 7.34; 0.25C₆H₁₄ N, 13.58 N, 13.23 56C₂₁H₂₈N₄O 349(+) C₂₁H₂₈N₄O C, 71.98; C, 72.07; 0.25CH₂Cl₂ H, 7.40; H,7.12; 0.08C₆H₁₄ N, 14.87 N, 14.70 57 C₁₇H_(14F)N₃O₂ 312(+) C₁₇H₁₄FN₃O₂C, 65.21; C, 65.10; 0.10H₂O H, 4.57; H, 4.48; N, 13.42 N, 13.25 58C₁₇H₁₄ClN₃O 328/ C₁₇H₁₄ClN₃O₂ C, 62.29; C, 62.33; 330(+) H, 4.31; H,4.24; N, 12.82 N, 12.70 59 C₁₇H₁₆ClN₃ 298/ C₁₇H₁₆ClN₃ C, 68.56; C,68.33; 300(+) H, 5.42; H, 5.40; N, 14.11 N, 13.91 60 C₂₀H₂₀FN₃ 322(+)C₂₀H₂₀FN₃ C, 74.97; C, 75.29; 0.10C₆H₁₄ H, 6.53; H, 6.30; N, 12.73 N,12.91 61 C₁₉H₂₂N₄ 307(+) C₁₉H₂₂N₄ C, 73.82; C, 73.78; 0.12CH₂Cl₂ H,7.45; H, 7.28; 0.06C₆H₁₄ N, 17.41 N, 17.07 62 C₁₇H₁₈N₄ 279(+) C₁₇H₁₈N₄C, 71.63; C, 71.75; 0.03CH₂Cl₂ H, 6.57; H, 6.52; 0.25C₆H₁₄ N, 19.15 N,18.94 0.30CH₄0 63 C₂₀H₂₂FN₃O 340(+) C₂₀H₂₂FN₃O C, 70.78; C, 70.71; H,6.53; H, 6.56; N, 12.38 N, 12.09 64 C₂₀H₂₀FN₃ 322(+) C₂₀H₂₀FN₃ C, 73.95;C, 73.89; 0.05CH₂Cl₂ H, 6.22; H, 6.22; N, 12.90 N, 12.58 65 C₁₇H₁₈N₄333(+) C₁₇H₁₈N₄ C, 75.88; C, 76.03; 0.32CH₂Cl₂ H, 7.92; H, 7.53;0.04C₆H₁₄ N, 15.42 N, 15.19 66 C₂₁H₂₀FN₃ 334(+) C₂₁H₂₀FN₃ C, 73.83; C,73.74; 0.12CH₂Cl₂ H, 5.94; H, 5.94; N, 12.23 N, 12.01 67 C₁₉H₁₈FN₃308(+) C₁₉H₁₈FN₃ C, 74.25; C, 74.19; H, 5.90; H, 6.01; N, 13.67 N, 13.3368 C₂₂H₂₄FN₃O 366(+) C₂₂H₂₄FN₃O 1.00HCl 69 C₂₆H₂₆FN₃ 400(+) C₂₆H₂₆FN₃1.00HCl 70 C₂₀H₂₂FN₃ 324(+) C₂₀H₂₂FN₃ 1.00HCl 71 C₂₅H₂₄FN₃ 386(+)C₂₅H₂₄FN₃ 1.00HCl 72 C₁₈H₁₈FN₃ 296(+) C₁₈H₁₈FN₃ C, 72.17; C, 72.30;0.14CH₂Cl₂ H, 6.44; H, 6.07; 0.05C₆H₁₄ N, 13.34 N, 12.95 73 C₁₇H₁₆ClN₃298/ C₁₇H₁₆ClN₃ C, 67.80; C, 67.76; 300(+) 0.10CH₂C₁₂ H, 5.65; H, 5.39;0.08C₆H₁₄ N, 13.42 N, 13.28 74 C₁₉H₂₀FN₃O 326(+) C₁₉H₂₀FN₃O C, 68.99; C,68.96; 0.08CH₂Cl₂ H, 6.12; H, 6.08; N, 12.65 N, 12.45 75 C₁₈H₁₈ClN₃ 312/C₁₈H₁₈ClN₃ C, 69.83; C, 70.16; 314(+) 0.13C₆H₁₄ H, 6.19; H, 5.88; N,13.01 N, 12.88 76 C₂₁H₂₄N₄O 349(+) C₂₁H₂₄N₄O C, 68.76; C, 68.30; H,6.85; H, 6.92; N, 14.65 N, 14.42 77 C₁₈H₁₇N₃O₂ 308(+) C₁₈H₁₇N₃O₂ C,66.28; C, 66.56; 0.22C₄H₈0₂ H, 5.59; H, 5.72; 0.23CH₂Cl₂ N, 12.14 N,11.75 78 C₂₀H₂₀BrN₃O₂ 414/ C₂₀H₂₀BrN₃O₂ C, 57.98; C, 57.79; 416(+) H,4.87; H, 4.90; N, 10.14 N, 9.99 79 C₁₇H₁₄FN₃O 296(+) C₁₇H₁₄FN₃O C,69.14; C, 69.05; H, 4.78; H, 4.90; N, 14.23 N, 13.89 80 C₂₀H₂₀BrN₃O₂414/ C₂₀H₂₀BrN₃O₂ C, 57.98; C, 58.16; 416(+) H, 4.87; H, 4.95; N, 10.14N, 10.16 81 C₂₀H₂₀ClN₃O₂ 370/ C₂₀H₂₀ClN₃O₂ C, 64.95; C, 65.03; 372(+) H,5.45; H, 5.54; N, 11.36 N, 11.37 82 C₂₀H₂₀ClN₃O₂ 370/ C₂₀H₂₀ClN₃O₂ C,62.72; C, 62.57; 372(+) 0.20CH₂Cl₂ H, 5.32; H, 5.39; N, 10.86 N, 10.7383 C₂₀H₂₀FN₃O₂ 354(+) C₂₀H₂₀FN₃O₂ C, 67.98; C, 67.87; H, 5.70; H, 5.73;N, 11.89 N, 11.67 84 C₂₄H₂₂FN₃O 388(+) C₂₄H₂₂FN₃O C, 73.75; C, 73.54;0.05CH₂Cl₂ H, 5.69; H, 5.80; N, 10.72 N, 10.62 85 C₂₃H₂₁FN₄ 373(+)C₂₃H₂₁FN₄ C, 73.82; C, 73.58; 0.10H₂O H, 5.71; H, 5.75; N, 14.97 N,14.91 86 C₁₉H₁₈FN₃O 324(+) C₁₉H₁₈FN₃O C, 64.02; C, 63.62; 0.50CH₂Cl₂ H,5.23; H, 5.26; N, 11.49 N, 11.49 87 C₂₂H₂₆N₄O 363(+) C₂₂H₂₆N₄O C, 73.61;C, 73.86; 0.30C₆H₁₄ H, 7.84; H, 7.53; N, 14.43 N, 14.57 88 C₁₈H₂₀N₄293(+) C₁₈H₂₀N₄ C, 73.86; C, 73.99; 0.03C₄H₈0₂ H, 6.99; H, 6.98;0.03C₆H₁₄ N, 18.83 N, 18.50 89 C₁₉H₁₈ClN₃ 324(+) C₁₉H₁₈ClN₃ C, 70.53; C,70.67; 0.02CH₂Cl₂ H, 5.86; H, 5.54; 0.08C₆H₁₄ N, 12.58 N, 12.23 90C₂₃H₂₆N₄O 375(+) C₂₃H₂₆N₄O C, 74.03; C, 73.88; 0.12C₆H₁₄ H, 7.25; H,7.19; N, 14.56 N, 14.24 91 C₂₂H₂₂FN₃O 364(+) C₂₂H₂₂FN₃O 0.10C₆H₁₄ 92C₂₁H₂₀FN₃O 350(+) C₂₁H₂₀FN₃O C, 71.69; C, 71.78; 0.15H₂O H, 5.86; H,5.67; 0.02C₆H₁₄ N, 11.87 N, 11.49 93 C₁₉H₁₈FN₃O 324(+) C₁₉H₁₈FN₃O C,70.57; C, 70.71; H, 5.61; H, 5.74; N, 12.99 N, 13.02 94 C₂₁H₂₂FN₃ 336(+)C₂₁H₂₂FN₃ C, 75.47; C, 75.68; 0.10C₆H₁₄ H, 6.85; H, 6.51; N, 12.28 N,12.28 95 C₂₃H₁₉F₂N₃ 376(+) C₂₃H₁₉F₂N₃ C, 72.89; C, 72.98; 0.20H₂O H,5.16; H, 5.13; N, 11.09 N, 10.92 96 C₂₄H₂₁ClFN₃ 406/ C₂₄H₂₁ClFN₃ C,71.02; C, 70.85; 408(+) H, 5.22; H, 5.25; N, 10.35 N, 10.17 97C₂₆H₂₁FN₄O₂ 441(+) C₂₆H₂₁FN₄O₂ 1.00HCl 98 C₂₃H₁₈Cl₂FN₃ 426/ C₂₃H₁₈Cl₂FN₃428(+) 2.00HCl 99 C₁₈H₁₆FN₃O 310(+) C₁₈H₁₆FN₃O C, 69.26; C, 69.12;0.15C₄H₈0₂ H, 5.38; H, 5.38; N, 13.03 N, 12.98 100 C₂₄H₂₁ClFN₃ 406/C₂₄H₂₁ClFN₃ 408(+) 1.00HCl 101 C₂₅H₂₄FN₃O₂ 418(+) 102 C₂₃H₁₈Cl₂FN₃ 426/428(+) 103 C₂₄H₂₁F₂N₃ 390(+) 104 C₂₄H₂₂FN₃ 372(+) 105 C₂₃H₁₉ClFN₃ 392/394(+) 106 C₂₅H₂₄FN₃ 386(+) C₂₅H₂₄FN₃ C, 75.28; C, 75.59; 0.04C₆H₁₄ H,6.71; H, 6.31; N, 12.40 N, 12.23 107 C₂₁H₂₂FN₃ 336(+) 108 C₂₀H₂₀FN₃322(+) 109 C₂₂H₂₄FN₃ 350(+) 110 C₂₄H₂₂FN₃ 336(+) 111 C₁₉H₂₀FN₃ 310(+)C₂₅H₂₄FN₃ C, 74.16; C, 74.54; 0.15C₆H₁₄ H, 6.91; H, 6.53; N, 13.04 N,13.23

1-9. (canceled)
 10. A compound of formula I,

wherein: R¹ is N(R⁴)₂, wherein R⁴ at each occurrence is independentlyselected from hydrogen, benzyl, C₁₋₆alkyl, C₃₋₇cycloalkyl, andmethoxyC₁₋₄alkyl; R² is N(R⁵)₂, wherein R⁵ at each occurrence isindependently selected from hydrogen, C₁₋₆alkyl, phenylC₁₋₆ alkyl,C₁₋₆alkenyl, C₃₋₇cycloalkyl, 1-methyl-C₃₋₇cycloal C₁₋₆alkylcarbonyl, andC₁₋₆alkoxycarbonyl; and R³ is phenyl substituted with E⁵, wherein E⁵ isselected from hydrogen, halogen, C₁₋₆alkoxy,NHC₁₋₃alkyl, andN(C₁₋₃alkyl)₂.
 11. A compound according to claim 10, wherein: R¹ isN(R⁴)₂, wherein R⁴ at each occurrence is independently selected fromhydrogen, benzyl, C₁₋₆alkyl, and methoxyC₁₋₄alkyl; R² is N(R⁵)₂, whereinR⁵ at each occurrence is independently selected from hydrogen,C₁₋₆alkyl, phenylC₁₋₆ alkyl, C₁₋₆alkenyl, C₃₋₇cycloalkyl, and1-methyl-C₃₋₇cycloalkyl; and R³ is phenyl substituted with E⁵, whereinE⁵ is selected from hydrogen, halogen, and C₁₋₆alkoxy.
 12. A compoundaccording to claim 10, wherein: R¹ is N(R⁴)₂, wherein R⁴ at eachoccurrence is independently hydrogen or C₁₋₆alkyl; R² is N(R⁵)₂, whereinR at each occurrence is independently hydrogen or C₁₋₆alkyl; and R³isphenyl substituted with E⁵, wherein E⁵ is halogen.
 13. A compoundaccording to claim 10, wherein R⁴ at each occurrence is independentlyhydrogen or C₁₋₆alkyl.
 14. A compound according to claim 13, wherein R⁴at one occurrence is hydrogen and at the other occurrence is C₁₋₆alkyl.15. A compound according to claim 10, wherein R⁵ at each occurrence isindependently hydrogen or C₁₋₆alkyl.
 16. A compound according to claim15, wherein R⁵ at one occurrence is hydrogen and at the other occurrenceis C₁₋₆alkyl.
 17. A compound according to claim 10, wherein R⁴ and R⁵ ateach occurrence are independently hydrogen or C₁₋₆alkyl.
 18. A compoundaccording to claim 17, wherein R⁴ and R⁵ at one occurrence are hydrogenand R⁴ and R⁵ at the other occurrence are C₁₋₆alkyl.
 19. A compoundaccording to claim 10, wherein E⁵is halogen.
 20. A compound according toclaim 19, wherein E⁵is fluoro.
 21. A compound according to claim 18,wherein E⁵ is fluoro.
 22. A compound according to claim 10, wherein R¹is NHCH₃, R² is NHCH₃, and E⁵ is fluoro.